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.

The Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS) - Part 2: Model sensitivity to the biomass burning

We describe an estimation technique for biomass burning emissions in South America based on a combination of remote-sensing fire products and field observations, the Brazilian Biomass Burning Emission Model (3BEM). For each fire pixel detected by remote sensing, the mass of the emitted tracer is calculated based on field observations of fire properties related to the type of vegetation burning. The burnt area is estimated from the instantaneous fire size retrieved by remote sensing, when available, or from statistical properties of the burn scars. The sources are then spatially and temporally distributed and assimilated daily by the Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS) in order to perform the prognosis of related tracer concentrations. Three other biomass burning inventories, including GFEDv2 and EDGAR, are simultaneously used to compare the emission strength in terms of the resultant tracer distribution. We also assess the effect of using the daily time resolution of fire emissions by including runs with monthly-averaged emissions. We evaluate the performance of the model using the different emission estimation techniques by comparing the model results with direct measurements of carbon monoxide both near-surface and airborne, as well as remote sensing derived products. The model results obtained using the 3BEM methodology of estimation introduced in this paper show relatively good agreement with the direct measurements and MOPITT data product, suggesting the reliability of the model at local to regional scales.

High p(T) direct photon and pi(0) triggered azimuthal jet correlations and measurement of k(T) for isolated direct photons in p plus p collisions at root s=200 GeV

Correlations of charged hadrons of 1< p(T) < 10 Gev/c with high pT direct photons and pi(0) mesons in the range 5< p(T) < 15 Gev/c are used to study jet fragmentation in the gamma + jet and dijet channels, respectively. The magnitude of the partonic transverse momentum, k(T), is obtained by comparing to a model incorporating a Gaussian kT smearing. The sensitivity of the associated charged hadron spectra to the underlying fragmentation function is tested and the data are compared to calculations using recent global fit results. The shape of the direct photon-associated hadron spectrum as well as its charge asymmetry are found to be consistent with a sample dominated by quark-gluon Compton scattering. No significant evidence of fragmentation photon correlated production is observed within experimental uncertainties.

Detailed measurement of the e(+)e(-) pair continuum in p plus p and Au plus Au collisions at root s(NN)=200 GeV and implications for direct photon production

PHENIX has measured the e(+)e(-) pair continuum in root s(NN) = 200 GeV Au+Au and p+p collisions over a wide range of mass and transverse momenta. The e(+)e(-) yield is compared to the expectations from hadronic sources, based on PHENIX measurements. In the intermediate-mass region, between the masses of the phi and the J/psi meson, the yield is consistent with expectations from correlated c (c) over bar production, although other mechanisms are not ruled out. In the low-mass region, below the phi, the p+p inclusive mass spectrum is well described by known contributions from light meson decays. In contrast, the Au+Au minimum bias inclusive mass spectrum in this region shows an enhancement by a factor of 4.7 +/- 0.4(stat) +/- 1.5(syst) +/- 0.9(model). At low mass (m(ee) < 0.3 GeV/c(2)) and high p(T) (1 < p(T) < 5 GeV/c) an enhanced e(+)e(-) pair yield is observed that is consistent with production of virtual direct photons. This excess is used to infer the yield of real direct photons. In central Au+Au collisions, the excess of the direct photon yield over the p+p is exponential in p(T), with inverse slope T = 221 +/- 19(stat) +/- 19(syst) MeV. Hydrodynamical models with initial temperatures ranging from T(init) similar or equal to 300-600 MeV at times of 0.6-0.15 fm/c after the collision are in qualitative agreement with the direct photon data in Au+Au. For low p(T) < 1 GeV/c the low-mass region shows a further significant enhancement that increases with centrality and has an inverse slope of T similar or equal to 100 MeV. Theoretical models underpredict the low-mass, low-p(T) enhancement.

Direct detection of Kaluza-Klein particles in neutrino telescopes

In theories with universal extra dimensions, all standard model fields propagate in the bulk and the lightest state of the first Kaluza-Klein (KK) level can be made stable by imposing a Z(2) parity. We consider a framework where the lightest KK particle (LKP) is a neutral, extremely weakly interacting particle such as the first KK excitation of the graviton, while the next-to-lightest KK particle (NLKP) is the first KK mode of a charged right-handed lepton. In such a scenario, due to its very small couplings to the LKP, the NLKP is long-lived. We investigate the production of these particles from the interaction of high energy neutrinos with nucleons in the Earth and determine the rate of NLKP events in neutrino telescopes. Using the Waxman-Bahcall limit for the neutrino flux, we find that the rate can be as large as a few hundreds of events a year for realistic values of the NLKP mass.

Intracellular mechanisms of specific beta-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure

beta-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (alpha(2A)/alpha(2C)-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca(2+)](i) transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca(2+)](i) transient, carvedilol had no effect on peak [Ca(2+)](i) transient but also increased [Ca(2+)] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca(2+) transients and the net balance of cardiac Ca(2+) handling proteins while carvedilol preferentially improved cardiac redox state. (C) 2008 Elsevier Inc. All rights reserved.

The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking alpha(2A) and alpha(2C) adrenoceptor knockout (alpha(2A)/alpha(2C) ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, alpha(2A)/alpha(2C)ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT(1) receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.

Coupled reliability and boundary element model for probabilistic fatigue life assessment in mixed mode crack propagation

Fatigue and crack propagation are phenomena affected by high uncertainties, where deterministic methods fail to predict accurately the structural life. The present work aims at coupling reliability analysis with boundary element method. The latter has been recognized as an accurate and efficient numerical technique to deal with mixed mode propagation, which is very interesting for reliability analysis. The coupled procedure allows us to consider uncertainties during the crack growth process. In addition, it computes the probability of fatigue failure for complex structural geometry and loading. Two coupling procedures are considered: direct coupling of reliability and mechanical solvers and indirect coupling by the response surface method. Numerical applications show the performance of the proposed models in lifetime assessment under uncertainties, where the direct method has shown faster convergence than response surface method. (C) 2010 Elsevier Ltd. All rights reserved.

Distributed power control algorithm for multiple access systems based on Verhulst model

In this paper the continuous Verhulst dynamic model is used to synthesize a new distributed power control algorithm (DPCA) for use in direct sequence code division multiple access (DS-CDMA) systems. The Verhulst model was initially designed to describe the population growth of biological species under food and physical space restrictions. The discretization of the corresponding differential equation is accomplished via the Euler numeric integration (ENI) method. Analytical convergence conditions for the proposed DPCA are also established. Several properties of the proposed recursive algorithm, such as Euclidean distance from optimum vector after convergence, convergence speed, normalized mean squared error (NSE), average power consumption per user, performance under dynamics channels, and implementation complexity aspects, are analyzed through simulations. The simulation results are compared with two other DPCAs: the classic algorithm derived by Foschini and Miljanic and the sigmoidal of Uykan and Koivo. Under estimated errors conditions, the proposed DPCA exhibits smaller discrepancy from the optimum power vector solution and better convergence (under fixed and adaptive convergence factor) than the classic and sigmoidal DPCAs. (C) 2010 Elsevier GmbH. All rights reserved.

Parametric correlation functions to model the structure of permanent environmental (co)variances in milk yield random regression models

The objective of the present study was to estimate milk yield genetic parameters applying random regression models and parametric correlation functions combined with a variance function to model animal permanent environmental effects. A total of 152,145 test-day milk yields from 7,317 first lactations of Holstein cows belonging to herds located in the southeastern region of Brazil were analyzed. Test-day milk yields were divided into 44 weekly classes of days in milk. Contemporary groups were defined by herd-test-day comprising a total of 2,539 classes. The model included direct additive genetic, permanent environmental, and residual random effects. The following fixed effects were considered: contemporary group, age of cow at calving (linear and quadratic regressions), and the population average lactation curve modeled by fourth-order orthogonal Legendre polynomial. Additive genetic effects were modeled by random regression on orthogonal Legendre polynomials of days in milk, whereas permanent environmental effects were estimated using a stationary or nonstationary parametric correlation function combined with a variance function of different orders. The structure of residual variances was modeled using a step function containing 6 variance classes. The genetic parameter estimates obtained with the model using a stationary correlation function associated with a variance function to model permanent environmental effects were similar to those obtained with models employing orthogonal Legendre polynomials for the same effect. A model using a sixth-order polynomial for additive effects and a stationary parametric correlation function associated with a seventh-order variance function to model permanent environmental effects would be sufficient for data fitting.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase AN INTEGRATED, ANIMATED MODEL

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na(+) and K(+) translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P(2c)-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E(1)/E(2)-ATPase as it undergoes conformational changes between the E(1) and E(2) forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger`s scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na(+) and K(+) translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the ""core engine"" of the pump, with respect to ATP binding, cation transport, and ADP and P(i) release.

Low Hematocrit Levels Increase Intracranial Pressure in an Animal Model of Cryogenic Brain Injury

Background: Brain injury is responsible for significant morbidity and mortality in trauma patients, but controversy still exists over optimal fluid management for these patients. This study aimed to investigate the effects of acute hemodilution with hydroxyethyl starch (HES) or lactated Ringer`s solution (LR) in intracranial pressure (ICP) and cerebral perfusion pressure (CPP) in dogs submitted to a cryogenic brain injury model. Methods: Design-Prospective laboratory animal study. Setting-Research laboratory in a teaching hospital. Subjects-Thirty-five male mongrel dogs. Interventions-Animals were enrolled to five groups: control, hemodilution with LR or HES 6% to an hematocrit target of 27% or 35%. Results: ICP and CPP levels were measured after cryogenic brain injury. Hemodilution promotes an increment of ICP levels, which decreases CPP when hematocrit target was estimated in 27.% after hemodilution. However, no differences were observed regarding crystalloid or colloid solution used for hemodilution in ICP and CPP levels. Conclusions: Hemodilution to a low hematocrit level increases ICP and decreases CPP scores in dogs submitted to a cryogenic brain injury. These results suggest that excessive hemodilution to a hematocrit below 30% should be avoided in traumatic brain injury patients.

Human breast tumor slices: A model for identification of vitamin D regulated genes in the tumor microenvironment

While many studies have addressed the direct effects of 1 alpha,25(OH)(2)D(3) on breast cancer (BC) cells, stromal-epithelial interactions, which are important for the tumor development, have been largely ignored. In addition, high concentrations of the hormone, which cannot be attained in vivo, have been used. Our aim was to establish a more physiological breast cancer model, represented by BC tissue slices, which maintain epithelial-mesenchymal interactions, cultured with a relatively low 1 alpha,25(OH)(2)D(3) concentration, in order to evaluate the vitamin D pathway. Freshly excised human BC samples were sliced and cultured in complete culture media containing vehicle, 0.5 nM or 100 nM 1 alpha,25(OH)(2)D(3) for 24 h. BC slices remained viable for at least 24 h, as evaluated by preserved tissue morphology in hematoxylin and eosin (HE) stained sections and bromodeoxyuridine (BrdU) incorporation by 10% of tumor cells. VDR mRNA expression was detected in all samples and CYP24A1 mRNA expression was induced by 1 alpha,25(OH)(2)D(3) in both concentrations (but mainly with 100 nM). Our results indicate that the vitamin D signaling pathway is functional in BC slices, a model which preserves stromal-epithelial interactions and mimics in vivo conditions. (C) 2010 Elsevier Ltd. All rights reserved.

Total Pancreatectomy: Porcine Model for Inducing Diabetes - Anatomical Assessment and Surgical Aspects

Background: The swine is an essential model for carrying out preclinical research and for teaching complex surgical procedures. There is a lack of experimental models describing anatomical and surgical aspects of total pancreatectomy in the pig. Materials and Methods: The experiments were performed on 10 white male swine weighing 27-33 kg. The animals were premedicated with midazolam (0.4 mg/kg, i.m.) and ketamine (4 mg/kg, i.m.). Anesthesia was induced with propofol (1-2 mg/kg, i.v.) and was maintained with propofol and fentanyl (0.3 mg and 0.1 mu g/kg/min, respectively, i.v.). The surgical period ranged from 44 to 77 min. The pancreas anatomy, and the main arterial, venous and pancreatic duct anatomy were assessed. Results: The pancreas anatomy was composed of 3 lobes, the `splenic`, `duodenal` and `connecting` lobe which is attached to the anterior portion of the portal vein. The splenic artery and the junction of the splenic vein and portal vein were divided. The left gastric artery was dissected and separated from its origin at the splenic artery. The head of the pancreas is disposed in a C shape. The pancreas was dissected and liberated from the right portion of the portal vein and the infrahepatic vena cava. The pancreas was separated from the duodenum preserving the pancreaticoduodenal artery, then we performed the total pancreatectomy preserving the duodenum, common bile duct and spleen. Conclusion: Total pancreatectomy with duodenum, bile duct and spleen preservation in the pig is feasible and an important instrument for research purposes and teaching surgical technique. Copyright (C) 2010 S. Karger AG, Basel

Role of cytokines in mediating mechanical hypernociception in a model of delayed-type hypersensitivity in mice

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF-alpha and IL-1 beta) and chemokines (KC/CXCL-1) in the genesis of mechanical hypernociception during antigen-induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand-held force transducer (electronic anesthesiometer) adapted with a 0.5 mm(2) polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham-immunized (SI) mice, induced mechanical hypernociception in a dose-dependant manner. Hypernociception induced by antigen was reduced in animals pretreated with IL-lra and reparixin (a non-competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1-/-) mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1 beta and KC/CXCL-1 in IM, but not in SI, mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1 beta and KC/CXCL-1 in IM, but not in SI, mice. The increase in TNF-alpha levels preceded the increase in IL-1 beta and KC/CXCL1. Antigen-induced release of IL-1 beta and KC/CXCL1 was reduced in TNFR1-/- mice, and TNF-alpha induced hypernociception was inhibited by IL-lra and reparixin. Hypernociception induced by IL-1 beta in immunized mice was inhibited by indomethacin, whereas KC/CXCL1-induced hypernociception was inhibited by indomethacin and guanethidine, Antigen-induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF-alpha, which triggers that subsequent release of IL-1 beta and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct-acting final mediator, prostanoids and sympathetic amines. (C) 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.