Large-volume sample stacking-capillary electrophoresis used for the determination of 3-nitrotyrosine in rat urine

Large-volume sample stacking using the electroosmotic flow (EOF) pump technique has been investigated for the quantification of 3-nitrotyrosine in urine of diabetic rats. The best separation conditions for these highly complex samples were obtained using capillary electrophoresis (CE) in the reversed polarity mode (i.e., injecting at the cathode and detecting at the anode) using cetyltrimethylammonium bromide (CTAB) in the running buffer. The optimum CE separation conditions were achieved using a phosphate buffer prepared with 0.15M phosphoric acid and 0.5 mM CTAB adjusted to pH 6.4 with sodium hydroxide. In such CE conditions, the limit of detection (LOD) was 1.77 microM for 3-nitrotyrosine with normal injection mode, meanwhile with the large-volume sample stacking technique a more than 20-fold improvement was observed (i.e., LOD = 0.08 microM was obtained) without noticeable loss of resolution. This value allowed the detection of 3-nitrotyrosine in urine from diabetic rats. To our knowledge, this work is one of the few applications showing the great possibilities of these stacking procedures to analyse biological samples by CE.     

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

BackgroundLeishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection.Conclusions/SignificanceDespite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the LbrPGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.     

Metabolites secreted by human atherothrombotic aneurysms revealed through a metabolomic approach

Abdominal aortic aneurysm (AAA) is perma-nent and localized dilation of the abdominal aorta. Intraluminal thrombus (ILT) is involved in evolution and rupture of AAA. Complex biological processes associated with AAA include oxidative stress, proteolysis, neovascularization, aortic inflammation, cell death, and extracellular matrix breakdown. Biomarkers of growth and AAA rupture could give a more nuanced indication for surgery, unveil novel pathogenic pathways, and open possibilities for pharmacological inhibition of growth. Differential analysis of metabolites released by normal and pathological arteries in culture may help to find molecules that have a high probability of later being found in plasma and start signaling processes or be useful diagnostic/prognostic markers. We used a LC-QTOF-MS metabolomic approach to analyze metabolites released by human ILT (divided into luminal and abluminal layers), aneurysm wall (AW), and healthy wall (HW). Statistical analysis was used to compare luminal with abluminal ILT layer, ILT with AW, and AW with HW to select the metabolites exchanged between tissue and external medium. Identified compounds are related to inflammation and oxidative stress and indicate the possible role of fatty acid amides in AAA. Some metabolites (e.g., hippuric acid) had not been previously associated to aneurysm, others (fatty acid amides) have arisen, indicating a very promising line of research.     

Bending continuous structures with SMAs: a novel robotic fish design

In this paper, we describe our research on bio-inspired locomotion systems using deformable structures and smart materials, concretely shape memory alloys (SMAs). These types of materials allow us to explore the possibility of building motor-less and gear-less robots. A swimming underwater fish-like robot has been developed whose movements are generated using SMAs. These actuators are suitable for bending the continuous backbone of the fish, which in turn causes a change in the curvature of the body. This type of structural arrangement is inspired by fish red muscles, which are mainly recruited during steady swimming for the bending of a flexible but nearly incompressible structure such as the fishbone. This paper reviews the design process of these bio-inspired structures, from the motivations and physiological inspiration to the mechatronics design, control and simulations, leading to actual experimental trials and results. The focus of this work is to present the mechanisms by which standard swimming patterns can be reproduced with the proposed design. Moreover, the performance of the SMA-based actuators' control in terms of actuation speed and position accuracy is also addressed.     

Stimulation characteristics that determine arteriolar dilation in skeletal muscle

To determine the skeletal muscle stimulation parameters that are most important in establishing vasodilation in the microvasculature, I tested whether arteriolar diameter during 2 min of repetitive, short-duration, tetanic skeletal muscle contractions increased with changes in stimulus frequency, stimulation train duration, and contraction frequency. To test this, the diameter of transverse arterioles approximately perpendicular to small bundles of cremaster muscle fibers in situ of anesthetized Golden Syrian hamsters was used as a bioassay system. Arteriolar diameter was measured before and during different stimulation patterns that consisted of a contraction frequency [6, 12, or 24 contractions per minute (cpm)], a stimulation train duration (250, 500, or 750 ms) and a stimulus frequency (4, 8, 10, 15, 20, 30, 40, 60, and 80 Hz). The magnitude of the dilation significantly increased with stimulus frequency but not in a simple linear manner. The average rate of increase was 0.32 +/- 0.02 microm/Hz from 4 to 20 Hz and 0.09 +/- 0.02 microm/Hz from 30 to 80 Hz. The magnitude of the dilation increased significantly with the contraction frequency where the dilation at 6 cpm was significantly smaller than the dilation at 24 cpm across all stimulus frequencies. Changing the train duration from 250 to 750 ms did not significantly affect the magnitude of the dilation. These observations suggest that stimulation parameters are important in determining the magnitude of the microvascular dilation and that the magnitude of the dilation was dependent on both the contraction frequency and stimulus frequency but was independent of train duration.     

Rapid biphasic arteriolar dilations induced by skeletal muscle contraction are dependent on stimulation characteristics

To test the hypothesis that measurable changes in microvasculature dilation occur in response to a single short-duration tetanic contraction, we contracted three to five skeletal muscle fibres of the hamster cremaster muscle microvascular preparation (in situ) and evaluated the response of an arteriole overlapping the active muscle fibres. Arteriolar diameter (baseline diameter = 16.4 +/- 0.9 micro m, maximum diameter = 34.7 +/- 1.2 micro m) was measured before and after a single contraction resulting from a range of stimulus frequencies (4, 10, 20, 30, 40, 60, and 80 Hz) within a 250- or 500-ms train. Four and 10 Hz produced a significant dilation at 2.9 +/- 0.4 and 6.5 +/- 2.8 s, respectively, within a 250-ms train and 3.0 +/- 0.2 and 6.1 +/- 1.3 s, respectively, within a 500-ms train. Biphasic dilations were observed within a 250-ms train at 20 Hz (at 3.9 +/- 0.9 and 22.1 +/- 4.3 s), 30 Hz (at 2.7 +/- 0.3 and 17.5 +/- 2.9 s), and 40 Hz (at 3.8 +/- 0.4 and 23.2 +/- 2.6 s) and within a 500-ms train at 20 Hz (at 4.8 +/- 0.4 and 31.9 +/- 3.8 s) and 30 Hz (at 3.4 +/- 0.3 and 27.6 +/- 3.0 s). A single dilation was observed within a 250-ms train at 60 Hz (at 5.1 +/- 0.7 s) and 80 Hz (at 14.2 +/- 3.3 s) and within a 500-ms train at 40 Hz (at 9.9 +/- 3.2 s), 60 Hz (at 7.9 +/- 2.1 s), and 80 Hz (at 13.4 +/- 4.0 s). We have shown that a single contraction ranging from a single twitch (4 Hz, 250 ms) to fused tetanic contractions produces significant arteriolar dilations and that the pattern of dilation is dependent on the stimulus frequency and train duration.     

An Improved Phenylarsine Oxide-Affinity Method Identifies Triose Phosphate Isomerase as a Candidate Redox Receptor Protein

Reversible oxidation on proteins of vicinal thiols to form intraprotein disulfides is believed to be an important means by which redox sensitivity is conferred on cellular signaling and metabolism. Affinity chromatography using immobilized phenylarsine oxide (PAO), which binds preferentially to vicinal thiols over monothiols, has been used in very limited studies to isolate the fraction of cellular proteins that exhibit reversible oxidation of vicinal thiols to presumed disulfide bonds. A challenge to the use of PAO-affinity chromatography for isolation of readily oxidizable vicinal thiol proteins (VTPs) has been the lack of a disulfide reducing agent that reverses oxidation of the PAO-binding protein thiols and maintains these in the reduced state necessary to bind PAO but does not also compete with the VTPs for binding to the immobilized PAO. The present study demonstrates that the capture from a detergent-soluble rat brain extract of VTPs by PAO-affinity chromatography was improved greatly by use of the reducing agent tris(2-carboxyethyl)-phosphine which, unlike more traditional disulfide-reducing agents, does not contain a thiol group. Moreover, we show that, while a substantial fraction of total brain proteins contain PAO-binding thiols, only a fraction of these were readily and reversibly oxidized. The two most abundant of these redox-active proteins were identified as albumin and triose phosphate isomerase (TPI). We propose that TPI is a candidate intracellular redox receptor protein. The improved PAO-affinity method detailed here should enable the discovery of lower abundance novel redox-active regulatory proteins.     

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.