A GDI/GDF-like system for sorting and shuttling ciliary proteins

Post/co-translational modifications by the addition of lipids take place in a vast number of proteins. Rab and Rho are small G proteins which are prenylated and targeted to membranes in complex with solubilizing factors called guanosine dissociation inhibitors (GDIs). The release of Rab and Rho at the correct destination from their cognate GDI has been proposed to be mediated through GDI displacement factors. However this mechanism is yet to be established and it has been shown that loading of Rab proteins with GTP at the destination can be sufficient for their correct targeting. PDE6D shares structural homology with Rho GDI and solubilises several prenylated proteins and mediate their targeting to different destinations including cilia. In a paper published by Fansa et al, the authors propose that sorting of cargo is dependent on the differential release by bona fide GDFs, Arl2 and Arl3, and the localization of the active Arl3GTP in cilia

Spatiotemporal regulation of signaling: focus on T cell activation and the immunological synapse

In a signaling network, not only the functions of molecules are important but when (temporal) and where (spatial) those functions are exerted and orchestrated is what defines the signaling output. To temporally and spatially modulate signaling events, cells generate specialized functional domains with variable lifetime and size that concentrate signaling molecules, enhancing their transduction potential. The plasma membrane is a key in this regulation, as it constitutes a primary signaling hub that integrates signals within and across the membrane. Here, we examine some of the mechanisms that cells exhibit to spatiotemporally regulate signal transduction, focusing on the early events of T cell activation from triggering of T cell receptor to formation and maturation of the immunological synapse

Engineering Metropolis: Contagion, Capital, and the Making of British Colonial Cairo, 1882-1922

This dissertation traces the transition of colonial Cairo from a marginal space to the British regime to an object of colonial governance and the site of technological and social intervention. It examines what caused this transition, how it shaped the spatial and social landscape of a booming metropolis, and how these developments produced and sustained opportunities, contradictions, and spaces for contestation and opposition. This dissertation challenges the current literature on British Cairo, which treats the colonial era (1882-1922) as a homogeneous expression of the regime’s retreat and of capital-led growth, by providing an account of the regime’s program of infrastructural reorganization and schemes of public housing and town planning. Because the literature largely ignores this history, it does not detect the colonial regime’s increasing discomfort at capital-led urban development or the regime’s late attempt to refashion its relation to capital and to take charge of Cairo’s future growth. The first part of this dissertation examines the pressures and crises that led to this transition. A protracted biological crisis that saw waves of cholera epidemics and high death rates underscored the need for constructing and improving infrastructures of sanitation and service provision. And capital’s forceful entry into the city led to a speculative property bubble, a housing crisis, and uncoordinated urban expansion, which made the disjointed framework of urban administration and the absence of regulations all the more evident. These crises made the colonial regime liable to critiques from elites, proponents, and certainly from the nascent anticolonial movement. The second part examines projects of sanitation and schemes of housing and town planning that the regime turned to since the beginning of the twentieth century and that embodied a changing approach to the city. During the latter two decades of the occupation, the colonial regime invested in upgrading Cairo’s water supply and constructing the city’s first sewage network. This dissertation traces not only how these infrastructural technologies worked but also how they became sites of contestation over power and knowledge. It examines the reception of infrastructures by urban dwellers across the social spectrum, the techno-social debates they occasioned among expert managers and designers, including above all engineers and public hygienists, and the social visions they embodied. Finally, the regime broached projects of public housing and town planning that constituted, in one sense, the culmination of a program of infrastructural reorganization, and in another, an attempt to give coherence to urban governance and assume leadership over the city’s development. By offering material improvement, these schemes were also meant to neutralize political discontent, which nonetheless erupted with the 1919 revolution

Experimental Characterization of Electrical Discharge Machining of Aluminum 6061 T6 Alloy using Different Dielectrics

Electrical discharge machining is a non-traditional machining method broadly employed in industries for machining of parts that have typical profiles and require great accuracy. This paper investigates the effects of electrical parameters: pulse-on-time and current on three performance measures (material removal rate, microstructures and electrode wear rate), using distilled water and kerosene as dielectrics. A comparison between dielectrics for the machining of aluminum 6061 T6 alloy material in terms of performance measures was performed. Aluminum 6061 T6 alloy material was selected, because of its growing use in the automotive and aerospace industrial sectors. The experimental sequence was designed using Taguchi technique of L9 orthogonal array by changing three levels of pulse-on-time and current, and test runs were performed separately for each dielectric. The results obtained show that greater electrode wear rate (EWR) and higher material removal rate (MRR) were achieved with distilled water when compared with kerosene. These greater EWR and MRR responses can be attributed to the early breakage of the weak oxide and carbide layers formed on the tool and alloy material surfaces, respectively. The innovative contributions of this study include, but are not limited to, the possibility of machining of aluminum 6061 T6 alloy with graphite electrode to enhance machinability and fast cutting rate employing two different dielectrics.Peer reviewe

PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

The phosphodiesterase 6 delta subunit (PDE6δ) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 5′-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the −1 and −3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destinati

The ciliary machinery is repurposed for T cell immune synapse trafficking of LCK

Upon engagement of the T cell receptor with an antigen-presenting cell, LCK initiates TCR signaling by phosphorylating its activation motifs. However, the mechanism of LCK activation specifically at the immune synapse is a major question. We show that phosphorylation of the LCK activating Y394, despite modestly increasing its catalytic rate, dramatically focuses LCK localization to the immune synapse. We describe a trafficking mechanism whereby UNC119A extracts membrane-bound LCK by sequestering the hydrophobic myristoyl group, followed by release at the target membrane under the control of the ciliary ARL3/ARL13B. The UNC119A N terminus acts as a “regulatory arm” by binding the LCK kinase domain, an interaction inhibited by LCK Y394 phosphorylation, thus together with the ARL3/ARL13B machinery ensuring immune synapse focusing of active LCK. We propose that the ciliary machinery has been repurposed by T cells to generate and maintain polarized segregation of signals such as activated LCK at the immune synapse

Multi-objective optimisation for minimum quantity lubrication assisted milling process based on hybrid response surface methodology and multi-objective genetic algorithm

© 2019 by SAGE Publications Ltd.Parametric modelling and optimisation play an important role in choosing the best or optimal cutting conditions and parameters during machining to achieve the desirable results. However, analysis of optimisation of minimum quantity lubrication–assisted milling process has not been addressed in detail. Minimum quantity lubrication method is very effective for cost reduction and promotes green machining. Hence, this article focuses on minimum quantity lubrication–assisted milling machining parameters on AISI 1045 material surface roughness and power consumption. A novel low-cost power measurement system is developed to measure the power consumption. A predictive mathematical model is developed for surface roughness and power consumption. The effects of minimum quantity lubrication and machining parameters are examined to determine the optimum conditions with minimum surface roughness and minimum power consumption. Empirical models are developed to predict surface roughness and power of machine tool effectively and accurately using response surface methodology and multi-objective optimisation genetic algorithm. Comparison of results obtained from response surface methodology and multi-objective optimisation genetic algorithm depict that both measured and predicted values have a close agreement. This model could be helpful to select the best combination of end-milling machining parameters to save power consumption and time, consequently, increasing both productivity and profitability.Peer reviewedFinal Published versio

A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB

Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form. This method incorporates a four step chromatography purification with TEV protease-mediated affinity tag cleavage and a conditioning step that achieves the activation of the GTPase by exchanging GDP for the non-hydrolyzable GTP analogue GMPPnP. We also demonstrate that an automated method is efficient at loading of KRas with mantGDP for application in a SOS1 catalysed fluorescent nucleotide exchange assay. In comparison to more conventional manual workflows the automated method offers marked advantages in method run time and operator workload. This reduces the bottleneck in protein production while generating products that are highly purified and effectively loaded with nucleotide analogues

Comprehensive Study on Tool Wear During Machining of Fibre-Reinforced Polymeric Composites

© 2021 Springer-Verlag. The final publication is available at Springer via https://dx.doi.org/10.1007/978-981-33-4153-1.The use of fibre reinforced polymeric (FRP) composites has increased rapidly, especially in many manufacturing (aerospace, automobile and construction) industries. The machining of composite materials is an important manufacturing process. It has attracted several studies over the last decades. Tool wear is a key factor that contributes to the cost of the machining process annually. It occurs due to sudden geometrical damage, frictional force and temperature rise at the tool-work interaction region. Moreover, tool wear is an inevitable, gradual and complex phenomenon. It often causes machined-induced damage on the workpiece/FRP composite materials. Considering the geometry of drill, tool wear may occur at the flank face, rake face and/or cutting edge. There are several factors affecting the tool wear. These include, but are not limited to, drilling parameters and environments/conditions, drill/tool materials and geometries, FRP composite compositions and machining techniques. Hence, this chapter focuses on drilling parameters, tool materials and geometries, drilling environments, types of tool wear, mechanisms of tool wear and methods of measurement of wear, effects of wear on machining of composite materials and preventive measures against rapid drill wear. Conclusively, some future perspectives or outlooks concerning the use of drill tools and their associated wears are elucidated, especially with the advancement in science and technology.Peer reviewedFinal Accepted Versio

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion