In vitro Evidence that an Aqueous Extract of Centella asiatica Modulates alpha-Synuclein Aggregation Dynamics

alpha-Synuclein aggregation is one of the major etiological factors implicated in Parkinson's disease (PD). The prevention of aggregation of alpha-synuclein is a potential therapeutic intervention for preventing PD. The discovery of natural products as alternative drugs to treat PD and related disorders is a current trend. The aqueous extract of Centella asiatica (CA) is traditionally used as a brain tonic and CA is known to improve cognition and memory. There are limited data on the role of CA in modulating amyloid-beta (A beta) levels in the brain and in A beta aggregation. Our study focuses on CA as a modulator of the alpha-synuclein aggregation pattern in vitro. Our investigation is focused on: (i) whether the CA leaf aqueous extract prevents the formation of aggregates from monomers (Phase I: alpha-synuclein + extract co-incubation); (ii) whether the CA aqueous extract prevents the formation of fibrils from oligomers (Phase II: extract added after oligomers formation); and (iii) whether the CA aqueous extract disintegrates the pre-formed fibrils (Phase III: extract added to mature fibrils and incubated for 9 days). The aggregation kinetics are studied using a thioflavin-T assay, circular dichroism, and transmission electron microscopy. The results showed that the CA aqueous extract completely inhibited the alpha-synuclein aggregation from monomers. Further, CA extract significantly inhibited the formation of oligomer to aggregates and favored the disintegration of the preformed fibrils. The study provides an insight in finding new natural products for future PD therapeutics.

Tripodal Bile Acid Architectures Based on a Triarylphosphine Oxide Core Obtained by Copper-Catalysed 1,3]-Dipolar Cycloaddition: Synthesis and Preliminary Aggregation Studies

We report the synthesis and aggregation behaviour of new water-soluble, bile acid derived tripodal architectures based on a core derived from triphenylphosphine oxide. We employed the well-established copper-catalysed 1,3]-dipolar cycloaddition (CuAAC) for the construction of these tripodal molecules. The aggregation behaviour of these molecules in aqueous media was studied by different analytical methods such as dye solubilisation, dynamic light scattering, NMR and AFM. These molecular architectures also offer an additional advantage in aiding understanding of the influence of the nature of the bile acid backbone and of the configuration at the steroid C-3 position in these architectures; to the best of our knowledge this has not been reported in the literature. The unique gelation properties of the -derivatives were explained through molecular modelling studies and the mechanical behaviour of these gels was studied by rheology experiments.

Fine-Tuning Dual Emission and Aggregation-Induced Emission Switching in NPI-BODIPY Dyads

Three new NPI-BODIPY dyads 1-3 (NPI = 1,8-naphthalimide, BODIPY = boron-dipyrromethene) were synthesized, characterized, and studied. The NPI and BODIPY moieties in these dyads are electronically separated by oxoaryl bridges, and the compounds only differ structurally with respect to methyl substituents on the BODIPY fluorophore. The NPI and BODIPY moieties retain their optical features in molecular dyads 1-3. Dyads 1-3 show dual emission in solution originating from the two separate fluorescent units. The variations of the dual emission in these compounds are controlled by the structural flexibilities of the systems. Dyads 13, depending on their molecular flexibilities, show considerably different spectral shapes and dissimilar intensity ratios of the two emission bands. The dyads also show significant aggregation-induced emission switching (AIES) on formation of nano-aggregates in THF/H2O with changes in emission color from green to red. Whereas the flexible and aggregation-prone compound 1 shows AIES, rigid systems with less favorable intermolecular interactions (i.e., 2 and 3) show aggregation-induced quenching of emission. Correlations of the emission intensity and structural flexibility were found to be reversed in solution and aggregated states. Photophysical and structural investigations suggested that intermolecular interactions (e. g., pi-pi stacking) play a major role in controlling the emission of these compounds in the aggregated state.

Experimental Investigation on Switching Characteristics of High-Current Insulated Gate Bipolar Transistors at Low Currents

Insulated gate bipolar transistors (IGBTs) are used in high-power voltage-source converters rated up to hundreds of kilowatts or even a few megawatts. Knowledge of device switching characteristics is required for reliable design and operation of the converters. Switching characteristics are studied widely at high current levels, and corresponding data are available in datasheets. But the devices in a converter also switch low currents close to the zero crossings of the line currents. Further, the switching behaviour under these conditions could significantly influence the output waveform quality including zero crossover distortion. Hence, the switching characteristics of high-current IGBTs (300-A and 75-A IGBT modules) at low load current magnitudes are investigated experimentally in this paper. The collector current, gate-emitter voltage and collector-emitter voltage are measured at various low values of current (less than 10% of the device rated current). A specially designed in-house constructed coaxial current transformer (CCT) is used for device current measurement without increasing the loop inductance in the power circuit. Experimental results show that the device voltage rise time increases significantly during turn-off transitions at low currents.

An Advanced Gate Driver for Insulated Gate Bipolar Transistors to Eliminate Dead-time Induced Distortions in Inverter Output

Dead-time is introduced between the gating signals to the top and bottom switches in a voltage source inverter (VSI) leg, to prevent shoot through fault due to the finite turn-off times of IGBTs. The dead-time results in a delay when the incoming device is an IGBT, resulting in error voltage pulses in the inverter output voltage. This paper presents the design, fabrication and testing of an advanced gate driver, which eliminates dead-time and consequent output distortion. Here, the gating pulses are generated such that the incoming IGBT transition is not delayed and shoot-through is also prevented. The various logic units of the driver card and fault tolerance of the driver are verified through extensive tests on different topologies such as chopper, half-bridge and full-bridge inverter, and also at different conditions of load. Experimental results demonstrate the improvement in the load current waveform quality with the proposed circuit, on account of elimination of dead-time.

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

Huntington's disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK. (C) 2014 Elsevier Inc. All rights reserved.

Suppression of protein aggregation by gold nanoparticles: a new way to store and transport proteins

Suppression of the aggregation of proteins has tremendous implications in biology and medicine. In the pharmaceuticals industry, aggregation of therapeutically important proteins and peptides while stored, reduces the efficacy and promptness of action leading to, in many instances, intoxication of the patient by the aggregate. Here we report the effect of gold nanoparticles (Au-NPs) in preventing the thermal and chemical aggregation of two unrelated proteins of different size, alcohol dehydrogenase (ADH, 84 kDa) and insulin (6 kDa), respectively, in physiological pH. Our principal observation is that there is a significant reduction (up to 95%) in the extent of aggregation of ADH and insulin in the presence of gold nanoparticles (Au-NPs). Aggregation of these proteins at micromolar concentration is prevented using nanomolar or less amounts of gold nanoparticles which is remarkable since chaperones which prevent such aggregation in vivo are required in micromolar quantity. The prevention of aggregation of these two different proteins under two different denaturing environments has established the role of Au-NPs as a protein aggregation prevention agent. The extent of prevention increases rapidly with the increase in the size of the gold nanoparticles. Protein molecules get physisorbed on the gold nanoparticle surface and thus become inaccessible by the denaturing agent in solution. This adsorption of proteins on AuNPs has been established by a variety of techniques and assays.

Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant alpha-Synuclein

Accumulating evidence suggests that deposition of neurotoxic a-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson's disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against a-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of a-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of a-synuclein amyloidosis and toxicity in Parkinson's disease and other synucleinopathies.

Experimental Investigation on Switching Characteristics of High-Current Insulated Gate Bipolar Transistors at Low Currents

Insulated gate bipolar transistors (IGBTs) are used in high-power voltage-source converters rated up to hundreds of kilowatts or even a few megawatts. Knowledge of device switching characteristics is required for reliable design and operation of the converters. Switching characteristics are studied widely at high current levels, and corresponding data are available in datasheets. But the devices in a converter also switch low currents close to the zero crossings of the line currents. Further, the switching behaviour under these conditions could significantly influence the output waveform quality including zero crossover distortion. Hence, the switching characteristics of high-current IGBTs (300-A and 75-A IGBT modules) at low load current magnitudes are investigated experimentally in this paper. The collector current, gate-emitter voltage and collector-emitter voltage are measured at various low values of current (less than 10% of the device rated current). A specially designed in-house constructed coaxial current transformer (CCT) is used for device current measurement without increasing the loop inductance in the power circuit. Experimental results show that the device voltage rise time increases significantly during turn-off transitions at low currents.

Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

Interferon-gamma (Ifn gamma), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifn gamma to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifn gamma, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/ nitric oxide adduct (DETA/NO), and Nos2(-/-) mice identified Nitric oxide (NO) induced by Ifn gamma as a key regulator of aggregation of APECs. Further studies with Nos2(-/-) APECs revealed that some Ifn. responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifn gamma and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifn gamma contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifn gamma and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or ``group behavior'' of APECs are discussed in the context of host resistance to infectious organisms.

Experimental characterisation and performance evaluation of a coaxial current transformer for measurement of insulated gate bipolar transistor switching current

Measurement of device current during switching characterisation of an insulated gate bipolar transistor (IGBT) requires a current sensor with low insertion impedance and high bandwidth. This study presents an experimental procedure for evaluating the performance of a coaxial current transformer (CCT), designed for the above purpose. A prototype CCT, which can be mounted directly on a power terminal of a 1200 V/50 A half-bridge IGBT module, is characterised experimentally. The measured characteristics include insertion impedance, gain and phase of the CCT at different frequencies. The bounds of linearity within which the CCT can operate without saturation are determined theoretically, and are also verified experimentally. The experimental study on linearity of the CCT requires a high-amplitude current source. A proportional-resonant (PR) controller-based current-controlled half-bridge inverter is developed for this purpose. A systematic procedure for selection of PR controller parameters is also reported in this study. This set-up is helpful to determine the limit of linearity and also to measure the frequency response of the CCT at realistic amplitudes of current in the low-frequency range.

Towards an Understanding of the Influence of Sedimentation on Colloidal Aggregation by Peclet Number

The Peclet number is a useful index to estimate the importance of sedimentation as compared to the Brownian motion. However, how to choose the characteristic length scale for the Peclet number evaluation is rather critical because the diffusion length increases as the square root of the time whereas the drifting length is linearly related to time. Our Brownian dynamics simulation shows that the degree of sedimentation influence on the coagulation decreases when the dispersion volume fraction increases. Therefore using a fixed length, such as the diameter of particle, as the characteristic length scale for Peclet number evaluation is not a good choice when dealing with the influence of sedimentation on coagulation. The simulations demonstrated that environmental factors in the coagulation process, such as dispersion volume fraction and size distribution, should be taken into account for more reasonable evaluation of the sedimentation influence.

Probing protein aggregation with quartz crystal microbalances.

The supra-molecular self-assembly of peptides and proteins is a process which underlies a range of normal and aberrant biological pathways in nature, but one which remains challenging to monitor in a quantitative way. We discuss the experimental details of an approach to this problem which involves the direct measurement in vitro of mass changes of the aggregates as new molecules attach to them. The required mass sensitivity can be achieved by the use of a quartz crystal transducer-based microbalance. The technique should be broadly applicable to the study of protein aggregation, as well as to the identification and characterisation of inhibitors and modulators of this process.

An In-Situ Observation On Initial Aggregation Process Of Colloidal Particles Near Three-Phase Contact Line Of Air, Water And Vertical Substrate

The self-assembling process near the three-phase contact line of air, water and vertical substrate is widely used to produce various kinds of nanostructured materials and devices. We perform an in-situ observation on the self-assembling process in the vicinity of the three phase contact line. Three kinds of aggregations, i.e. particle-particle aggregation, particle-chain aggregation and chain-chain aggregation, in the initial stage of vertical deposition process are revealed by our experiments. It is found that the particle particle aggregation and the particle-chain aggregation can be qualitatively explained by the theory of the capillary immersion force and mirror image force, while the chain-chain aggregation leaves an opening question for the further studies. The present study may provide more deep insight into the self-assembling process of colloidal particles.