Severe congenital neutropenia (SCN) is characterized by a deficiency of mature neutrophils, leading to recurrent bacterial and fungal infections. Although mutations in Elastase-2, neutrophil (ELA2) predominate in human SCN, mutation of Ela2 in mice does not recapitulate SCN. The growth factor independent-1 (GFI1) transcription factor regulates ELA2. Mutations in GFI1 are associated with human SCN, and genetic deletion of Gfi1 results in murine neutropenia. We examined whether human SCN-associated GFI1N382S mutant proteins are causal in SCN and found that GFI1 functions as a rate-limiting granulopoietic molecular switch. The N382S mutation inhibited GFI1 DNA binding and resulted in a dominant-negative block to murine granulopoiesis. Moreover, Gfi1N382S selectively derepressed the monopoietic cytokine CSF1 and its receptor. Gfi1N382S-expressing Csf1-/- cells formed neutrophils. These results reveal a common transcriptional program that underlies both human and murine myelopoiesis, and that is central to the pathogenesis of SCN associated with mutations in GFI1. This shared transcriptional pathway may provide new avenues for understanding SCN caused by mutations in other genes and for clinical intervention into human neutropenias.
Seven transmembrane receptors widely known as G-proteincoupled receptors (GPCRs) 4 (1, 2) mediate an array of physiological processes in response to such diverse agonists as peptides, amino acid derivatives, and lipids. Despite the great diversity in their ligands, the conserved motifs found across this superfamily and the limited interacting partners such as G-proteins (3) and -arrestins (4) at the cytoplasmic interface point toward a common activation mechanism for GPCRs. GPCRs constitute the single largest group of molecules for drug targets due to their critical importance in mediating biological responses as well as their easy accessibility on the cell surface. However, very little structural information is available for GPCRs due to difficulties in purifying and obtaining crystal structures for this class of receptors. The availability of the rhodopsin crystal structure (5) combined with the approach of computational modeling and validation by site-directed mutagenesis has led to delineation of ligand-receptor interactions in a few GPCRs (6 -9). Some elements of the activation mechanism have been identified for individual GPCRs (1,6,8,[10][11][12]. Several studies employing site-directed mutagenesis have helped uncover critical interactions between residues in transmembrane domains of the GPCRs (reviewed in Ref. 1). The approach of computational modeling with validation by site-directed mutagenesis has led to significant increases in the understanding of the processes involved in GPCR activation (6 -9).Leukotriene B 4 (LTB 4 ) is a potent leukocyte chemoattractant and mediates its biological effects through two distinct GPCRs, the high affinity receptor BLT1 and the low affinity receptor BLT2 (13,14). Several recent studies suggested a direct and critical role for BLT1 in diverse inflammatory diseases such as arthritis (15, 16), atherosclerosis (17, 18), and asthma (19). Recently, the high affinity LTB 4 receptor, BLT1, was expressed in Escherichia coli and shown to form a functional pentameric complex with heterotrimeric G-proteins (20). Computational modeling has been used to investigate the potential role of the eighth helix in signaling of BLT1 (21,22). In addition, a recent study reported an LTB 4 binding site in BLT1 deduced from computational models (23). However, the exact nature of the LTB 4 binding site and the potential changes in receptor conformation following LTB 4 binding remain unknown.In this study, computational modeling together with sitedirected mutagenesis led to precise mapping and validation of the LTB 4 binding site in BLT1. Mutation of each of the residues predicted to be in the putative binding site resulted in reduced binding affinity. Furthermore, analysis of dynamic structures of the ligand-free and ligand-bound BLT1 allowed prediction of critical movements of transmembrane helices and essential
Efficient and cost optimal feature of microfluidic based biochips are inspiring in automation of clinical diagnostics. Many laboratory oriented biochemical tests are transfigured into on chip operations. Droplet based microfluidic is a sub-category of microfluidic sciences. Actually this type of fluidic device is capable of manipulating multiple droplets concurrently. The main aim of droplet routing in a Digital Microfluidic Biochip (DMFB) is to find an efficient path for each droplet from a designated source electrode to a destination electrode under different fluidic and static constraints of microfluidic operations. The whole operation can be make more scalable and controlled through a programmable intelligent controller. In order to match up with the current needs and required flexibility of commercial applications, this intelligent controller has to take care of many issues like pin optimization, cross reference minimization and contamination detection during routing. This makes the task of intelligent controller quite complex and challenging. These online controllers need to control multiple biochip boards at different time intervals to promote optimum resource and time utilization. However, online controllers are very susceptible to wide range of cyber attacks. In recent literature, varieties of cyber physical attacks have been reported which cause remarkable impairment on the operation of physical system. This proposed method is able to adjudge such malicious operations at the physical system level. This will help to monitor the behavior of the physical operation and can stall it under any abnormality in operation. Experimental findings show that the proposed technique can detect the errors and superfluous operations accurately with minimum consumption of computing resources. Comparative performance of error detection efficiency shows betterment over the existing methods.
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