Using multidimensional projection techniques for reaching a high distinguishing ability in biosensing

Recent advances in the control of molecular engineering architectures have allowed unprecedented ability of molecular recognition in biosensing, with a promising impact for clinical diagnosis and environment control. The availability of large amounts of data from electrical, optical, or electrochemical measurements requires, however, sophisticated data treatment in order to optimize sensing performance. In this study, we show how an information visualization system based on projections, referred to as Projection Explorer (PEx), can be used to achieve high performance for biosensors made with nanostructured films containing immobilized antigens. As a proof of concept, various visualizations were obtained with impedance spectroscopy data from an array of sensors whose electrical response could be specific toward a given antibody (analyte) owing to molecular recognition processes. In addition to discussing the distinct methods for projection and normalization of the data, we demonstrate that an excellent distinction can be made between real samples tested positive for Chagas disease and Leishmaniasis, which could not be achieved with conventional statistical methods. Such high performance probably arose from the possibility of treating the data in the whole frequency range. Through a systematic analysis, it was inferred that Sammon`s mapping with standardization to normalize the data gives the best results, where distinction could be made of blood serum samples containing 10(-7) mg/mL of the antibody. The method inherent in PEx and the procedures for analyzing the impedance data are entirely generic and can be extended to optimize any type of sensor or biosensor.

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

Parametric analyses of anxiety in zebrafish scototaxis

Scototaxis, the preference for dark environments in detriment of bright ones, is an index of anxiety in zebrafish. In this work, we analyzed avoidance of the white compartment by analysis of the spatiotemporal pattern of exploratory behavior (time spent in the white compartment of the apparatus and shuttle frequency between compartments) and swimming ethogram (thigmotaxis, freezing and burst swimming in the white compartment) in four experiments. In Experiment 1, we demonstrate that spatiotemporal measures of white avoidance and locomotion do not habituate during a single 15-min session. In Experiments 2 and 3, we demonstrate that locomotor activity habituates to repeated exposures to the apparatus, regardless of whether inter-trial interval is 15-min or 24-h; however, no habituation of white avoidance was observed in either experiment. In Experiment 4, we confined animals for three 15-min sessions in the white compartment prior to recording spatiotemporal and ethogram measures in a standard preference test. After these forced exposures, white avoidance and locomotor activity showed no differences in relation to non-confined animals, but burst swimming, thigmotaxis and freezing in the white compartment were all decreased. These results suggest that neither avoidance of the white compartment nor approach to the black compartment account for the behavior of zebrafish in the scototaxis test. (C) 2010 Elsevier B.V. All rights reserved.

The unconditioned fear produced by morphine withdrawal is regulated by mu- and kappa-opioid receptors in the midbrain tectum

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures-the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala(2) N-Me-Phe(4) Gly(5)-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 mu l) - a selective mu-receptor agonist - or nor-binaltorphimine (BNI; 2.5 and 5 mu g/0.2 mu l) - a selective K-receptor antagonist with tardive action - on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region. (C) 2009 Elsevier B.V. All rights reserved.

Involvement of the midbrain tectum in the unconditioned fear promoted by morphine withdrawal

The midbrain rectum structures, dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), are involved in the organization of fear and anxiety states during the exposure to dangerous stimuli. Since opiate withdrawal is associated with increased anxiety in both humans and animals, this study aimed to investigate the possible sensitization of the neural substrates of fear in the midbrain tectum and its influence on the morphine withdrawal-induced anxiety. For the production of drug withdrawal, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of the chronic treatment, independent groups were probed in the elevated plus-maze and open-field tests. Additional groups of animals were implanted with a bipolar electrode into the dPAG OF the IC and submitted to the electrical stimulation of these structures for the determination of the freezing and escape thresholds after 48 h of withdrawal. Our results showed that the morphine withdrawal promoted clear-cut levels of anxiety without the somatic signs of opiate withdrawal. Moreover, morphine-withdrawn rats had an increase in the reactivity to the electrical stimulation of the dPAG and the IC. These findings suggest that the increased anxiety induced by morphine withdrawal is associated with the sensitization of the neural substrates of fear in the dPAG and the IC. So, the present results give support to the hypothesis that withdrawal from chronic treatment with morphine leads to fear states possibly engendered by activation of the dPAG and IC, regardless of the production of somatic symptoms. (C) 2008 Elsevier B.V. All rights reserved.

Midazolam reduces the selective activation of the rhinal cortex by contextual fear stimuli

Independent brain circuits appear to underlie different forms of conditioned fear, depending on the type of conditioning used, such as a context or explicit cue paired with footshocks. Several clinical reports have associated damage to the medial temporal lobe (MTL) with retrograde amnesia. Although a number of studies have elucidated the neural circuits underlying conditioned fear, the involvement of MTL components in the aversive conditioning paradigm is still unclear. To address this issue, we assessed freezing responses and Fos protein expression in subregions of the rhinal cortex and ventral hippocampus of rats following exposure to a context, light or tone previously paired with footshock (Experiment 1). A comparable degree of freezing was observed in the three types of conditioned fear, but with distinct patterns of Fos distribution. The groups exposed to cued fear conditioning did not show changes in Fos expression, whereas the group subjected to contextual fear conditioning showed selective activation of the ectorhinal (Ect), perirhinal (Per), and entorhinal (Ent) cortices, with no changes in the ventral hippocampus. We then examined the effects of the benzodiazepine midazolam injected bilaterally into these three rhinal subregions in the expression of contextual fear conditioning (Experiment 2). Midazolam administration into the Ect, Per, and Ent reduced freezing responses. These findings suggest that contextual and explicit stimuli endowed with aversive properties through conditioning recruit distinct brain areas, and the rhinal cortex appears to be critical for storing context-, but not explicit cue-footshock, associations. (C) 2010 Elsevier B.V. All rights reserved.

Serotonergic mechanisms of the median raphe nucleus-dorsal hippocampus in conditioned fear: Output circuit involves the prefrontal cortex and amygdala

Independent studies have shown that the median raphe nucleus (MRN) and dorsal hippocampus (DH) are involved in the expression of contextual conditioned fear (CFC). However, studies that examine the integrated involvement of serotonergic mechanisms of the MRN-DH are lacking. To address this issue, a CFC paradigm was used to test whether the serotonergic projections from the MRN to DH can influence CFC. Serotoninergic drugs were infused either into the MRN or DH prior to testing sessions in which freezing and startle responses were measured in the same context where 6 h previously rats received footshocks. A reduction of serotonin (5-HT) transmission in the MRN by local infusions of the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) decreased freezing in response to the context but did not reduce fear-potentiated startle. This pattern of results is consistent with the hypothesis that MRN serotonergic mechanisms selectively modulate the freezing response to the aversive context. As for the DH, a decrease in postsynaptic 5-HT receptor activity at projection areas has been proposed to be the main consequence of 5-HT(1A) receptor activation in the MIRN. Intra-DH injections of 8-OH-DPAT inhibited both the freezing and fear-potentiated startle response to the context. To reconcile these findings, an inhibitory mechanism may exist between the incoming 5-HT pathway from the MRN to DH and the neurons of the DH output to other structures. The DH-amygdala or medial prefrontal cortex projections could well be this output circuit modulating the expression of CFC as revealed by measurements of Fos immunoreactivity in these areas. (C) 2009 Elsevier B.V. All rights reserved.

Temporal and spatial distribution of anurans in the Pampa Region (Santa Maria, RS).

The present study aimed to determine the richness, occurrence constancy, reproductive modes. standard of abundance distribution, season of vocalization and to test correlation among climatic variables and activity of vocalization of anurans in a region of the Pampa Biome, Santa Maria, Rio Grande do Sul State. During the period of Novernber/2001 to October/2002 monthly collections were carried out utilizing the `survey at breeding site` method and examination of specimens kept in the Colecao Herpetologica do Setor de Zoologia da Universidade Federal de Santa Maria (ZUFSM). Tire Occurrence of 25 species of anurans was recorded. The anurofauna recorded represents 30% of the species known to Occur in Rio Grande do Sul, and comprises species generally associated with grasslands in this state and neighboring countries. Four reproductive modes were recorded: mode 1 (14 species: 58.3%) mode 11 and 30 (9 species` 37.5%) and mode 24 (1 species; 4.2%). The low diversification of reproductive modes is likely related to the homogeneity of the grassland habitat. Most species were constant or accessory in the Study area and the species abundance distribution patterns fit in the Broken Stick and Log-normal models. characterized by homogeneity of species abundance distribution. Most species showed great plasticity in habitat. but few were plastic in vocalization sites use. There was a weak positive correlation between species richness and precipitation. There was also a weak positive correlation between the abundance of species calling activity and maximum average temperatures. These correlations indicated that, in the study area. the abundance of calling males is more affected by the temperature, and species richness is more affected by precipitation, despite the fact that significantly higher species richness occurs during the hottest period of the year. These results showed that the climatological variables examined were not enough to explain the seasonal occurrence of species, thus the influence of other environmental variables merit to be tested in future studies.

Tooth loss in Brazilian middle-aged adults: multilevel effects

Objective. To examine the link between tooth loss and multilevel factors in a national sample of middle-aged adults in Brazil. Material and methods. Analyses were based on the 2003 cross-sectional national epidemiological survey of the oral health of the Brazilian population, which covered 13 431 individuals (age 35-44 years). Multistage cluster sampling was used. The dependent variable was tooth loss and the independent variables were classified according to the individual or contextual level. A multilevel negative binomial regression model was adopted. Results. The average tooth loss was 14 (standard deviation 9.5) teeth. Half of the individuals had lost 12 teeth. The contextual variables showed independent effects on tooth loss. It was found that having 9 years or more of schooling was associated with protection against tooth loss (means ratio range 0.68-0.76). Not having visited the dentist and not having visited in the last >= 3 years accounted for increases of 33.5% and 21.3%, respectively, in the risk of tooth loss (P < 0.05). The increase in tooth extraction ratio showed a strong contextual effect on increased risk of tooth loss, besides changing the effect of protective variables. Conclusions. Tooth loss in middle-aged adults has important associations with social determinants of health. This study points to the importance of the social context as the main cause of oral health injuries suffered by most middle-aged Brazilian adults.

Chronic absence of baroreceptor inputs prevents training-induced cardiovascular adjustments in normotensive and spontaneously hypertensive rats

We investigate whether arterial baroreceptors mediate the training-induced blood pressure fall and resting bradycardia in hypertensive (SHR) and normotensive rats (WKY). Male SHR and WKY rats, submitted to sino-aortic denervation (SAD) or sham surgery (SHAM group), were allocated to training (T; 55% of maximal exercise capacity) or sedentary (S) protocols for 3 months. Rats were instrumented with arterial and venous catheters for haemodynamic measurements at rest (power spectral analysis) and baroreceptor testing. Kidney and skeletal muscles were processed for morphometric analysis of arterioles. Elevated mean arterial pressure (MAP) and heart rate (HR) in SHAM SHRS were accompanied by increased sympathetic variability and arteriolar wall/lumen ratio [+3.4-fold on low-frequency (LF) power and +70%, respectively, versus WKYS, P < 0.05]. Training caused significant HR (similar to 9% in WKY and SHR) and MAP reductions (-8% in the SHR), simultaneously with improvement of baroreceptor reflex control of HR (SHR and WKY), LF reduction (with a positive correlation between LF power and MAP levels in the SHR) and normalization of wall/lumen ratio of the skeletal muscle arterioles (SHR only). In contrast, SAD increased pressure variability in both strains of rats, causing reductions in MAP (-13%) and arteriolar wall/lumen ratio (-35%) only in the SHRS. Training effects were completely blocked by SAD in both strains; in addition, after SAD the resting MAP and HR and the wall/lumen ratio of skeletal muscle arterioles were higher in SHRT versus SHRS and similar to those of SHAM SHRS. The lack of training-induced effects in the chronic absence of baroreceptor inputs strongly suggests that baroreceptor signalling plays a decisive role in driving beneficial training-induced cardiovascular adjustments.

The systematic use of intraoperative vascular Doppler ultrasound during microsurgical subinguinal varicocelectomy improves precise identification and preservation of testicular blood supply

Objective: To evaluate the impact of systematic use of intraoperative Doppler ultrasound during microsurgical subinguinal varicocele repair. Design: Prospective clinical study. Setting: Andrology laboratory and male infertility section of the urology department of a tertiary care hospital. Patient(s): Two hundred and thirteen men with clinical varicocele. Intervention(s): Subinguinal microsurgical varicocele ligation using an intraoperative vascular Doppler flow detector. Main Outcome Measure(s): Number of veins ligated, lymphatic spared, arteries identified or accidentally ligated. Result(s): A statistically significant greater number of arteries were identified and preserved when intraoperative vascular Doppler was used. In addition, the average number of internal spermatic veins ligated was statistically significantly greater in the same group. Accidental artery ligation occurred in two cases (1.1%) in which the Doppler was not applied. There was no statistically significant difference in number of lymphatics spared between groups. Conclusion(s): Our findings showed that concomitant use of intraoperative vascular Doppler during microsurgical varicocelectomy allows more arterial branches to be preserved, and more internal spermatic veins are likely to be ligated. This device should be considered an attractive tool to improve surgical outcomes and safety. (Fertil Steril (R) 2010; 93: 2396-9. (C)2010 by American Society for Reproductive Medicine.)

Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia

Background. Vascular calcification (VC) is commonly seen in patients with chronic kidney disease (CKD). Elevated levels of phosphate and parathormone (PTH) are considered nontraditional risk factors for VC. It has been shown that, in vitro, phosphate transforms vascular smooth muscle cells (VSMCs) into calcifying cells, evidenced by upregulated expression of runt-related transcription factor 2 (Runx2), whereas PTH is protective against VC. In addition, Runx2 has been detected in calcified arteries of CKD patients. However, the in vivo effect of phosphate and PTH on Runx2 expression remains unknown. Methods. Wistar rats were submitted to parathyroidectomy, 5/6 nephrectomy (Nx) and continuous infusion of 1-34 rat PTH (at physiological or supraphysiological rates) or were sham-operated. Diets varied only in phosphate content, which was low (0.2%) or high (1.2%). Biochemical, histological, immunohistochemistry and immunofluorescence analyses were performed. Results. Nephrectomized animals receiving high-PTH infusion presented VC, regardless of the phosphate intake level. However, phosphate overload and normal PTH infusion induced phenotypic changes in VSMCs, as evidenced by upregulated aortic expression of Runx2. High-PTH infusion promoted histological changes in the expression of osteoprotegerin and type I collagen in calcified arteries. Conclusions. Phosphate, by itself is a potential pathogenic factor for VC. It is of note that phosphate overload, even without VC, was associated with overexpression of Runx2 in VSMCs. The mineral imbalance often seen in patients with CKD should be corrected.

Antioxidant Activity of Uruguayan Propolis. In Vitro and Cellular Assays

The antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 mu mol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol content (determined by Folin-Ciocalteu method) and UV absorption. Total polyphenol content (150 mg gallic acid equiv/g propolis) and flavonoids (45 mg quercetin equiv/g propolis) were similar to values reported for southern Brazilian (group 3) and Argentinean propolis. Flavonoid composition determined by RP-HPLC indicates a strong poplar-tree origin. Samples high in polyphenols efficiently inhibit low-density lipoprotein lipoperoxidation and tyrosine nitration. In addition, Uruguayan propolis was found to induce the expression of endothelial nitric oxide synthase and inhibit endothelial NADPH oxidase, suggesting a potential cardiovascular benefit by increasing nitric oxide bioavailability in the endothelium.

Smart Alerts: Development of Software to Optimize Data Monitoring

Dherte PM, Negrao MPG, Mori Neto S, Holzhacker R, Shimada V, Taberner P, Carmona MJC - Smart Alerts: Development of a Software to Optimize Data Monitoring. Background and objectives: Monitoring is useful for vital follow-ups and prevention, diagnosis, and treatment of several events in anesthesia. Although alarms can be useful in monitoring they can cause dangerous user`s desensitization. The objective of this study was to describe the development of specific software to integrate intraoperative monitoring parameters generating ""smart alerts"" that can help decision making, besides indicating possible diagnosis and treatment. Methods: A system that allowed flexibility in the definition of alerts, combining individual alarms of the parameters monitored to generate a more elaborated alert system was designed. After investigating a set of smart alerts, considered relevant in the surgical environment, a prototype was designed and evaluated, and additional suggestions were implemented in the final product. To verify the occurrence of smart alerts, the system underwent testing with data previously obtained during intraoperative monitoring of 64 patients. The system allows continuous analysis of monitored parameters, verifying the occurrence of smart alerts defined in the user interface. Results: With this system a potential 92% reduction in alarms was observed. We observed that in most situations that did not generate alerts individual alarms did not represent risk to the patient. Conclusions: Implementation of software can allow integration of the data monitored and generate information, such as possible diagnosis or interventions. An expressive potential reduction in the amount of alarms during surgery was observed. Information displayed by the system can be oftentimes more useful than analysis of isolated parameters.