Quantitative Imaging of Protein-Protein Interactions by Multiphoton Fluorescence Lifetime Imaging Microscopy using a Streak camera

Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.Comment: Overview of FLIM techniques, StreakFLIM instrument, FRET application

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

Defects in Friction Stir Welding of Steel

Defects associated with friction stir welding of two steel grades including DH36 and EH46 were investigated. Different welding parameters including tool rotational and tool traverse (linear) speeds were applied to understand their effect on weld seam defects including microcracks and voids formation. SEM images and infinite focus microscopy were employed to identify the defects types. Two new defects associated with the friction stir welding process are introduced in this work. The first defect identified in this work is a microcrack found between the plunge and the steady state region and attributed to the traverse moving of the tool with unsuitable speed from the plunge-dwell to the steady state stage. The tool traverse speed has recommended to travel 20 mm more with accelerated velocity range of 0.1 from the maximum traverse speed until reaching the steady state. The maximum recommended traverse speed in the steady state was also suggested to be less than 400 mm/min in order to avoid the lack in material flow. The second type of defect observed in this work was microcracks inside the stirred zone caused by elemental precipitations of TiN. The precipitates of TiN were attributed to the high tool rotational speed which caused the peak temperature to exceed 1200 °C at the top of the stirred zone and based on previous work. The limit of tool rotational speed was recommended to be maintained in the range of 200-500 RPM based on the mechanical experiments on the FSW samples

GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [^3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [^3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ~30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst

The Effect of Fluorine Doping on the Characteristic Behaviour of CdTe

Fluorine which is an n-type dopant of cadmium telluride thin films was included during growth from a cadmium nitrate [Cd(NO3)2Æ4H2O] bath using an electrodeposition technique. The fluorine concentration in the Cd(NO3)2Æ4H2O baths were varied between 0.5 ppm and 50 ppm in order to determine its effect on the optical, structural, morphological and electrical properties of the as-deposited and the post-growth-treated layers. These characterisations were carried out using x-ray diffraction, ultraviolet–visible spectrophotometry, scanning electron microscopy, energy dispersive x-rays spectroscopy, photoelectrochemical cell measurement, direct current conductivity measurement and fully fabricated device characterisation. The results are systematically reported in this paper

Temporal Resolution In Preschool Children

The purpose of this study was to examine temporal resolution in normal hearing pre-school children. Word recognition was evaluated in quiet and in spectrally identical continuous and interrupted noise at signal-to-noise ratios (S/Ns) of 10, 0, and -10 dB. Sixteen children four to five years of age and eight adults participated. Performance decreased with decreasing S/N. At poorer S/Ns, participants demonstrated superior performance or a release from masking in the interrupted noise. Adults performed better than children, yet, the release from masking was equivalent. Collectively these findings are consistent with the notion that preschool children suffer from poorer processing efficiency rather than temporal resolution per se

Improvement of Grazing Lands for Better Livestock Production--A Case Study from Chitradurga District in India

Chitradurga is a highly drought prone district in the central dry zone of Karnataka, India, with a normal rainfall of 530 mm per annum. Over 85 per cent of cultivable area is rainfed and the livestock plays a vital role in rural income generation in this district. Improper management and overgrazing have resulted in most of the grazing resources declining to a poor, degraded condition. Regeneration of pasture land was vital in the villages due to three reasons - a) people\u27s livelihood dependency on livestock was considerable b) small ruminants played a vital role for landless farmers and c) lack of adequate fodder was a prime factor for low livestock productivity. To improve the livelihood of livestock farmers, a few interventions were made under World Bank funded National Agricultural Innovation Project through consortium approach in 10 project villages. Frequent interactions were held with the local livestock farmers to discuss about the importance of increasing the fodder resources in the villages to improve the income and to sustain their livelihood. They realized the importance of fodder and came forward to take up cultivation of perennial fodders and also, for the revitalization of grazing lands, locally known as kavals. The primary survey in the villages indicated that about 90 to 96 per cent of the small ruminant holders are dependent on these common property grazing resources for the fodder needs. The High Level Panel of Experts on food security and nutrition, constituted by FAO, has emphasized the importance of extending appropriate technologies and inputs, providing the needed credit and ensuring assured and remunerative marketing opportunities to the smallholders (HLPE, 2013). Such measures are also essential for revitalizing the degraded grasslands in this region

Comparative Study of Ram Air Turbines based on Wind Tunnel Study for Specific Air Borne Energy Extraction

Ram Air Turbines (RAT) are used for emergency on-board power generation on aircraft and associated systems. Many studies on usage of RATs have shown promising results in terms of using RATs as a source of emergency on-board power generation. Many external podded systems on aircraft utilise RATs for self-sufficient adaptation. These pods generate their own power using RATs for their power requirements instead of depending on the mother aircraft power. Commercial cargo planes use RATs for generating emergency hydraulic power. A RAT was suggested to be used for emergency power, during failure of main alternator on a prototype aircraft. A specific requirement of the RAT was also to produce high drag for aerodynamic braking when deployed and concurrently generate electrical energy. Three models with different solidity were studied in wind tunnel at different wind speeds for suitability of this drag-energy combination. This paper presents the results of the study. Based on the results, a suitable RAT was selected for further analysis and ground trials

Musical Training Influences Auditory Temporal Processing

Background: A link between musical expertise and auditory temporal processing abilities was examined. Material and methods: Trained musicians (n=13) and non-musicians (n=12) were tested on speech tasks (phonetic identification, speech recognition in noise) and non-speech tasks (temporal gap detection). Results: Results indicated musicians had shorter between-channel gap detection thresholds and sharper phonetic identification functions, suggesting that perceptual reorganization following musical training assists basic temporal auditory processes. Conclusions: In general, our results provide a conceptual advance in understanding how musical training influences speech processing, an ability which, when impaired, can affect speech and reading competency