Adenylyl cyclase 5/6 underlie PIP3 dependent regulation

A wide variety of signaling substances such as hormones, neurotransmitters, odorants and chemokines control intracellular signaling by regulating the production of the second messenger cAMP. By activating Epac, PKA and cyclic nucleotide-gated ion channels, the production of cAMP alters a wide range of biological processes including cell division and metabolism. A number of GPCRs controls intracellular cAMP levels via stimulatory or inhibitory G proteins via adenylyl cyclases. The function of the broadly expressed AC5 and AC6 is enhanced by stimulatory (Gαs) or attenuated by inhibitory (Gαi) G proteins. Mechanistically both inhibition and stimulation is mediated via a direct protein-protein interaction. In addition to this direct regulation, several previous studies reported a cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes for which the mechanism is debated (Hartzell, 1988; Wang & Lipsius, 1995). A similar cAMP rebound response was observed previously in our lab after termination of α2A-AR adrenergic receptor activation in HEK293T cells (Markus et al., 2013). The present study was aimed at investigating mechanisms underlying Gi-induced cAMP rebound effects. Many genetically encoded biosensors have been developed based on fluorescence resonance energy transfer (FRET) to visualize the spatiotemporal dynamics of various intracellular signals including second messengers. FRET-based cAMP biosensor (Epac1-camps) as well as heterologous overexpression system was used to investigate the mechanisms underlying Gi-mediated cAMP rebound stimulation in cardiac myocytes and also in heterologous expression system. When studying the mechanism of the long-known phenomenon of cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes, we observed a PTX-sensitive/Gi-mediated/ adenylyl cyclase (type 5/6)/ cAMP-dependent pathway for this cAMP rebound stimulation. In addition, we observed that inhibition of Gβγ by gallein led to an attenuation of the AC5- mediated cAMP rebound response, although, overexpression of AC4 did not produce additional cAMP stimulation. This implies that different Gβγ-mediated signaling pathways may exist. Interestingly, we observed that PI3K inhibitor attenuates AC5/6-dependent cAMP rebound effects. This indicated that Gi-mediated cAMP rebound response was mediated via the PI3K-dependent pathway. Indeed, overexpression of PIP3-specific phosphatase PTEN confirmed that PIP3 itself either directly or indirectly mediated Gi-dependent cAMP rebound responses. Additionally, inhibition of PIP2-specific phosphatase SHIP and downstream events of PIP3-dependent regulation of Akt further confirm the influence of PIP3 on cAMP rebound levels. Indeed, surpassing Gi-mediated PI3K activation through PDGF-receptor stimulation strengthens this pathway. In addition, we confirmed that inhibition of PI3K also prevented cAMP rebound response after withdrawal of ACh in atrial myocytes. We suppose that the novel PIP3 dependent regulation of AC5/6 might represent a missing mechanism that explains physiological phenomena such as post vagal tachycardia

Securing Web Applications from malware attacks using hybrid feature extraction

In this technological era, many of the applications are taking the utilization of services of internet in order to cater to the needs of its users. With the rise in number of internet users, there's a substantial inflation within the internet attacks. Because of this hike, Web Services give rise to new security threats. One among the major concerns is the susceptibility of the internet services for cross site scripting (XSS). More than three fourths of the malicious attacks are contributed by XSS. This article primarily focuses on detection and exploiting XSS vulnerabilities. Generally, improper sanitization of input results in these type of susceptibilities. This article primarily focuses on fuzzing, and brute forcing parameters for XSS vulnerability. In addition, we've mentioned the planned framework for contradicting XSS vulnerability

Securing Web Applications from malware attacks using hybrid feature extraction

In this technological era, many of the applications are taking the utilization of services of internet in order to cater to the needs of its users. With the rise in number of internet users, there's a substantial inflation within the internet attacks. Because of this hike, Web Services give rise to new security threats. One among the major concerns is the susceptibility of the internet services for cross site scripting (XSS). More than three fourths of the malicious attacks are contributed by XSS. This article primarily focuses on detection and exploiting XSS vulnerabilities. Generally, improper sanitization of input results in these type of susceptibilities. This article primarily focuses on fuzzing, and brute forcing parameters for XSS vulnerability. In addition, we've mentioned the planned framework for contradicting XSS vulnerability

Numerical Simulation of Bi-component Droplet Evaporation and Dispersion in Spray and Spray Drying

Spray drying is one of the most widely used drying techniques to convert liquid feed into a dry powder. The modeling of spray flows and spray drying has been studied for many years now, to determine the characteristics of the end products, e.g. particle size, shape, density or porosity. So far, the simulation of polymer or sugar solution spray drying has not been studied because drying behavior as well as properties are unknown. Previous studies concentrated on the systems of milk, salt solution, colloids or other materials for which the thermal and physical properties are well tabulated. The present study deals with the modeling and simulation of polyvinylpyrrolidone (PVP)/water and mannitol/water spray flows. PVP is a polymer, widely used as a pharmaceutical excipient, and mainly manufactured by BASF under several patented names, whereas mannitol is a sugar, which is used in dry powder inhalers and tablets. Experimental studies have shown that the powder properties of PVP and mannitol are significantly influenced by the drying conditions. The growing importance of PVP or mannitol powders and the inability of existing studies to predict the effect of drying conditions on the properties of the end product have prompted the development of a new reliable model and numerical techniques. Evaporating sprays have a continuous phase (gas) and a dispersed phase, which consists of droplets of various sizes that may evaporate, coalesce, or breakup, as well as have their own inertia and size-conditioned dynamics. A modeling approach which is more commonly used is the Lagrangian description of the dispersed liquid phase. This approach gives detailed information on the micro-level, but inclusion of droplet coalescence and breakup increase computational complexity. Moreover, the Lagrangian description coupled with the Eulerian equations for the gas phase, assuming a point-source approximation of the spray, is computationally expensive. As an alternative to Lagrangian simulations, several Eulerian methods have been developed based on the Williams’ spray equation. The Euler – Euler methods are computationally efficient and independent of liquid mass loading in describing dense turbulent spray flows. The objective of this thesis is the modeling and simulation of spray flows and spray drying up to the onset of solid layer formation in an Euler – Euler framework. The behavior of droplet distribution under various drying conditions in bi-component evaporating spray flows is examined using, for the first time, direct quadrature method of moments (DQMOM) in two dimensions. In DQMOM, the droplet size and velocity distribution of the spray is modeled by approximating the number density function in terms of joint radius and velocity. Transport equations of DQMOM account for droplet evaporation, heating, drag, and droplet–droplet interactions. At first, an evaporating water spray in nitrogen is modeled in one dimension (axial direction). Earlier studies in spray flows neglected evaporation or considered it through a simplified model, which is addressed by implementing an advanced droplet evaporation model of Abramzon and Sirignano, whereas droplet motion and droplet coalescence are estimated through appropriate sub-models. The assumption of evaporative flux to be zero or computing it with weight ratio constraints was found to be unphysical, which is improved by estimating it using the maximum entropy formulation. The gas phase is not yet fully coupled to the DQMOM but its inlet properties are taken to compute forces acting on droplets and evaporation. The simulation results are compared with quadrature method of moments (QMOM) and with experiment at various cross sections. DQMOM shows better results than QMOM, and remarkable agreement with experiment. Next, water spray in air in two-dimensional, axisymmetric configuration is modeled by extending the one-dimensional DQMOM. The DQMOM results are compared with those of the discrete droplet model (DDM), which is an Euler – Lagrangian approach. Droplet coalescence is considered in DQMOM but neglected in DDM. The simulation results are validated with new experimental data. Overall, DQMOM shows a much better performance with respect to computational effort, even with the inclusion of droplet coalescence. Before extending DQMOM to model PVP/water spray flows, a single droplet evaporation and drying model is developed, because most of the evaporation models available in the literature are valid for salts, colloids or milk powder. The negligence of solid layer formation effects on the droplet heating and evaporation is addressed, and treatment of the liquid mixture as the ideal solution is improved by including the non-ideality effect. The PVP or mannitol in water droplet evaporation and solid layer formation are simulated, and the results are compared with new experimental data, which shows that the present model effectively captures the first three stages of evaporation and drying of a bi-component droplet. Finally, PVP/water spray flows in air are simulated using DQMOM including the developed bi-component evaporation model. Simulation results are compared with new experimental data at various cross sections and very good agreement is observed. In conclusion, water and PVP/water evaporating spray flows, and preliminary stages of PVP/water and mannitol/water spray drying, i.e., until solid layer formation, are successfully modeled and simulated, and show good agreement with experiment

Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes.

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10-15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes

Investigation of mass discharge rate and segregation from hopper by discrete element method

Hoppers of different shape and angle are widely used in different industries particularly in handling of solids as storage units and in unit operations, e.g. mixing, tableting, etc. It is a challenge to choose a right hopper to achieve desired flow and insignificant segregation due to difference in material properties. General approach for the selection of optimum hopper for a given unit operation is based on the trial-and-error experimental approach. To address this optimum hopper selection, combined experimental and numerical approach is presented in this study. The objective of this study is to analyze the effect of mixture composition and hopper angle on the flow rate and segregation behavior. The numerical simulation of granular flow out of various conical hoppers was also performed using the discrete element method (DEM). The materials considered include different particle size glass bead particles in different proportions by mass. The experimental study is done to validate the DEM results, particularly, mass flow rate. The results analyzed include temporal development of mass fraction of a given particle size during discharge. In addition, the mass flow rate is also computed. The results indicate that fines percentage in the mixture, ratio of smallest particle size to largest in the mixture, and hopper angle plays significant role in determining the segregation and mass flow rate. The flow pattern found to be influenced by the hopper angle and mean particle size of mixture. The results of discharge rate from DEM are also compared with existing empirical correlations and finite element method based elastoplastic model. The DEM prediction shows a good agreement with the existing correlations for a wide range of hopper angles, and with the experimental data

Numerical investigation of screw design influence on screw feeding in a roller compactor

Roller compaction refers to a dry granulation process where fine particulate feed is fed to the counter rotating rolls of a roller compactor to form ribbons which are further milled to produce free flowing agglomerates. For the continuous production of ribbons, there needs to be an adequate supply of powder by the screw to the rolls without any interruptions. In general, screws used in roller compactors are designed to convey powders of all types (cohesive, bulky, compressible, etc.), whereby usage of different screw designs for different powder types may be avoided. However, using such single screw type roller compactors for poor flowing powders may be challenging. On the other hand, the selection of the right screw for a given powder can only be done based on a combination of prior experience and trial-and-error experimentation. Empirical correlations exist to predict the draw down rate of screw feeders depending on their design, however, these correlations assume that there is continuous supply of powder by the screw, which limits its application to free-flowing powders only. To address this, in this study numerical simulations are performed based on discrete element method (DEM) to investigate the impact of screw design on the powder supply to rolls for cohesive and poorly flowing powders. The geometry considered includes a hopper, horizontal feeding screw below the hopper, and two counter-rotating rolls at the end of the screw. Two different screw designs are investigated where the main difference between them is the pitch length. The influence of scraper speed is investigated. Additionally, the influence of material attribute such as cohesion is studied. For both designs, the simulation results calculated include the rate of powder supply by the screw, velocity of particles in the screw etc. The simulation results of powder supply rate are also compared with results obtained based on empirical correlation. Overall, this simulation approach helps in selecting appropriate screw design for the given cohesive powder

Trace metals in vegetables and fruits cultivated around the surroundings of Tummalapalle uranium mining site, Andhra Pradesh, India

AbstractVegetables (Tomato – Solanum lycopersicum, green chilli – Capsicum annum and bitter gourd – Momordica charantia) and fruits (Banana – Musa acuminata colla, papaya – Carica papaya and mosambi – Citrus limetta) from the cultivated areas around the Tummalapalle uranium mining site were analyzed for trace metals (Al, Cr, Mn, Fe, Ni, Cu, Zn, Pb, Be, V, Co, Cd and U) using inductively coupled plasma-mass spectrometer (ICP-MS). As per the estimated data, the concentrations of trace metals in vegetables and fruits are found in the range of 47.5–7.8 mg/kg for Al, 9.7–1.0 mg/kg for Cr, 3.8–1.0 mg/kg for Mn, 75.5–13.9 mg/kg for Fe, 1.4–0.2 mg/kg for Ni, 2.3–0.8 mg/kg for Cu, 9.2–3.1 mg/kg for Zn, 0.2–1.4 mg/kg for Pb, 19.2–1.9 μg/kg for Be, 96.1–15.8 μg/kg for V, 48.2–12.9 μg/kg for Co, 46.5–2.3 μg/kg for Cd and 16.4–2.7 μg/kg for U. The trace metals observed are compared to the literature reported values. Trace elemental data were subjected to statistical analysis to examine the interrelationship between the investigated trace elements and possible source identification of the trace metal contamination in vegetable and fruits. Daily intake of trace metals through ingestion of vegetables and fruits are also calculated

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation

Development and characterisation of pressed packaging solutions for high-temperature high-reliability SiC power modules

SiC is a wide bandgap semiconductor with better electrothermal properties than silicon, including higher temperature of operation, higher breakdown voltage, lower losses and the ability to switch at higher frequencies. However, the power cycling performance of SiC devices in traditional silicon packaging systems is in need of further investigation since initial studies have shown reduced reliability. These traditional packaging systems have been developed for silicon, a semiconductor with different electrothermal and thermomechanical properties from SiC, hence the stresses on the different components of the package will change. Pressure packages, a packaging alternative where the weak elements of the traditional systems like wirebonds are removed, have demonstrated enhanced reliability for silicon devices however, there has not been much investigation on the performance of SiC devices in press-pack assemblies. This will be important for high power applications where reliability is critical. In this paper, SiC Schottky diodes in pressure packages have been evaluated, including the electrothermal characterisation for different clamping forces and contact materials, the thermal impedance evaluation and initial thermal cycling studies, focusing on the use of aluminium graphite as contact material