A novel neural network model with distributed evolutionary approach for big data classification.

The considerable improvement of technology produced for various applications has resulted in a growth in data sizes, such as healthcare data, which is renowned for having a large number of variables and data samples. Artificial neural networks (ANN) have demonstrated adaptability and effectiveness in classification, regression, and function approximation tasks. ANN is used extensively in function approximation, prediction, and classification. Irrespective of the task, ANN learns from the data by adjusting the edge weights to minimize the error between the actual and predicted values. Back Propagation is the most frequent learning technique that is used to learn the weights of ANN. However, this approach is prone to the problem of sluggish convergence, which is especially problematic in the case of Big Data. In this paper, we propose a Distributed Genetic Algorithm based ANN Learning Algorithm for addressing challenges associated with ANN learning for Big data. Genetic Algorithm is one of the well-utilized bio-inspired combinatorial optimization methods. Also, it is possible to parallelize it at multiple stages, and this may be done in an extremely effective manner for the distributed learning process. The proposed model is tested with various datasets to evaluate its realizability and efficiency. The results obtained from the experiments show that after a specific volume of data, the proposed learning method outperformed the traditional methods in terms of convergence time and accuracy. The proposed model outperformed the traditional model by almost 80% improvement in computational time.

Cloud vendor selection for the healthcare industry using a big data-driven decision model with probabilistic linguistic information.

The role of cloud services in the data-intensive industry is indispensable. Cision recently reported that the cloud market would grow to 55 billion USD, with an active contribution of the cloud to healthcare around 2025. Inspired by the report, cloud vendors expand their market and the quality of services to seek growth globally. The rapid growth of the cloud sector in the healthcare industry imposes a challenge: making a rational choice of a cloud vendor (CV) out of a diverse set of vendors. Typically, the healthcare industry 4.0 sees the issue as a large-scale group decision-making problem. Previous studies on a CV selection face certain challenges, such as (i) a lack of the ability to handle multiple users' views, as well as experts'/users' complex linguistic views; (ii) the confidence level associated with a view is not considered; (iii) the transformation of multiple users' views into holistic data is lacking; and (iv) the systematic prioritization of CVs with minimum human intervention is a crucial task. Motivated by these challenges and circumventing them, a new big data-driven decision model is put forward in this paper. Initially, the data in the form of complex expressions are collected from multiple cloud users and are further transformed into a holistic decision matrix by adopting probabilistic linguistic information (PLI). PLI represents complex linguistic expressions along with the associated confidence levels. Later, a holistic decision matrix is formed with the missing values imputed by proposing an imputation algorithm. Furthermore, the criteria weights are determined by using a newly proposed mathematical model and partial information. Finally, the evaluation based on the distance from average solution (EDAS) approach is extended to PLI for the rational ranking of CVs. A real-time example of a CV selection for a healthcare center in India is exemplified so as to demonstrate the usefulness of the model, and the comparison reveals the merits and limitations of the model.

An extended fuzzy decision-making framework using hesitant fuzzy sets for the drug selection to treat the mild symptoms of Coronavirus Disease 2019 (COVID-19).

The whole world is presently under threat from Coronavirus Disease 2019 (COVID-19), a new disease spread by a virus of the corona family, called a novel coronavirus. To date, the cases due to this disease are increasing exponentially, but there is no vaccine of COVID-19 available commercially. However, several antiviral therapies are used to treat the mild symptoms of COVID-19 disease. Still, it is quite complicated and uncertain decision to choose the best antiviral therapy to treat the mild symptom of COVID-19. Hesitant Fuzzy Sets (HFSs) are proven effective and valuable structures to express uncertain information in real-world issues. Therefore, here we used the hesitant fuzzy decision-making (DM) method. This study has chosen five methods or medicines to treat the mild symptom of COVID-19. These alternatives have been ranked by seven criteria for choosing an optimal method. The purpose of this study is to develop an innovative Additive Ratio Assessment (ARAS) approach to elucidate the DM problems. Next, a divergence measure based procedure is developed to assess the relative importance of the criteria rationally. To do this, a novel divergence measure is introduced for HFSs. A case study of drug selection for COVID-19 disease is considered to demonstrate the practicability and efficacy of the developed idea in real-life applications. Afterward, the outcome shows that Remdesivir is the best medicine for patients with mild symptoms of the COVID-19. Sensitivity analysis is presented to ensure the permanence of the introduced framework. Moreover, a comprehensive comparison with existing models is discussed to show the advantages of the developed framework. Finally, the results prove that the introduced ARAS approach is more effective and reliable than the existing models.

Predictive Analysis on Behavioural Risk Factor Surveillance System through Ensemble Classifiers and Clusters.

INTRODUCTION: The World Health Organization (WHO) has been asserting the importance of health care in today's world. The objective of this research is to find out the type of medication that needs to be provided to the people at early stages to prevent behavioural risk factors. The health department of United States has a great vision to improve the immune system of the people and has taken measures to do the same through a Behavioural Risk Factor Surveillance System (BRFSS). This research aims to prevent behavioural risk factors by predictive analysis using the above mentioned dataset from the Centres for Disease Control and Prevention (CDC). METHODOLOGY: The methods of ensemble classification and clustering is applied on the dataset, pre and post weighted classification, thereby classifying and prescribing the type of healthcare required for people exhibiting behaviours such as obesity, nutrition and physical activity. RESULTS AND DISCUSSION: This analyses help improve the quality of health of the citizens. In an extensive study, it was observed that the result obtained was 92.87% accurate.

Novel Approaches for Diagnosing Melanoma Skin Lesions Through Supervised and Deep Learning Algorithms.

Dermoscopy is a technique used to capture the images of skin, and these images are useful to analyze the different types of skin diseases. Malignant melanoma is a kind of skin cancer whose severity even leads to death. Earlier detection of melanoma prevents death and the clinicians can treat the patients to increase the chances of survival. Only few machine learning algorithms are developed to detect the melanoma using its features. This paper proposes a Computer Aided Diagnosis (CAD) system which equips efficient algorithms to classify and predict the melanoma. Enhancement of the images are done using Contrast Limited Adaptive Histogram Equalization technique (CLAHE) and median filter. A new segmentation algorithm called Normalized Otsu's Segmentation (NOS) is implemented to segment the affected skin lesion from the normal skin, which overcomes the problem of variable illumination. Fifteen features are derived and extracted from the segmented images are fed into the proposed classification techniques like Deep Learning based Neural Networks and Hybrid Adaboost-Support Vector Machine (SVM) algorithms. The proposed system is tested and validated with nearly 992 images (malignant & benign lesions) and it provides a high classification accuracy of 93 %. The proposed CAD system can assist the dermatologists to confirm the decision of the diagnosis and to avoid excisional biopsies.

Do not let death do us part: 'find-me' signals in communication between dying cells and the phagocytes.

The turnover and clearance of cells is an essential process that is part of many physiological and pathological processes. Improper or deficient clearance of apoptotic cells can lead to excessive inflammation and autoimmune disease. The steps involved in cell clearance include: migration of the phagocyte toward the proximity of the dying cells, specific recognition and internalization of the dying cell, and degradation of the corpse. The ability of phagocytes to recognize and react to dying cells to perform efficient and immunologically silent engulfment has been well-characterized in vitro and in vivo. However, how apoptotic cells themselves initiate the corpse removal and also influence the cells within the neighboring environment during clearance was less understood. Recent exciting observations suggest that apoptotic cells can attract phagocytes through the regulated release of 'find-me' signals. More recent studies also suggest that these find-me signals can have additional roles outside of phagocyte attraction to help orchestrate engulfment. This review will discuss our current understanding of the different find-me signals released by apoptotic cells, how they may be relevant in vivo, and their additional roles in facilitating engulfment.

An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response.

Cells undergo programmed cell death/apoptosis throughout the lifespan of an organism. The subsequent immunologically silent removal of apoptotic cells plays a role in the maintenance of tolerance; defects in corpse clearance have been associated with autoimmune disease. A number of receptors and signaling molecules involved in this process have been identified, but intracellular signaling downstream of corpse recognition is only now being defined. Calcium plays a key role as a second messenger in many cell types, leading to the activation of downstream molecules and eventual transcription of effector genes; however, the role of calcium signaling during apoptotic cell removal is unclear. Here, using studies in cell lines and in the context of a whole organism, we show that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that the genes required for calcium flux are essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium through CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in Caenorhabditis elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes seemed to affect early signaling events, leading to a decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the secretion of TGF-beta by the phagocytes during the engulfment of apoptotic cells. Taken together, these data point to an earlier unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: the formation of the phagocytic cup and the internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.

A multi-objective evolutionary algorithm for protein structure prediction with immune operators.

Genetic algorithms (GA) are often well suited for optimisation problems involving several conflicting objectives. It is more suitable to model the protein structure prediction problem as a multi-objective optimisation problem since the potential energy functions used in the literature to evaluate the conformation of a protein are based on the calculations of two different interaction energies: local (bond atoms) and non-local (non-bond atoms) and experiments have shown that those types of interactions are in conflict, by using the potential energy function, Chemistry at Harvard Macromolecular Mechanics. In this paper, we have modified the immune inspired Pareto archived evolutionary strategy (I-PAES) algorithm and denoted it as MI-PAES. It can effectively exploit some prior knowledge about the hydrophobic interactions, which is one of the most important driving forces in protein folding to make vaccines. The proposed MI-PAES is comparable with other evolutionary algorithms proposed in literature, both in terms of best solution found and the computational time and often results in much better search ability than that of the canonical GA.

Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells.

Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles. Finally, we identify MIG-2/RhoG (which functions upstream of Dock180 : ELMO) as a possible point of crosstalk between these two signaling modules. Taken together, these data suggest that Dock180/ELMO and CrkII act as two evolutionarily conserved signaling submodules that coordinately regulate engulfment.

CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration.

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Signaling via Shc family adapter proteins.

The adapter protein Shc was initially identified as an SH2 containing proto-oncogene involved in growth factor signaling. Since then a number of studies in multiple systems have implicated a role for Shc in signaling via many different types of receptors, such as growth factor receptors, antigen receptors, cytokine receptors, G-protein coupled receptors, hormone receptors and integrins. In addition to the ubiquitous ShcA, two other shc gene products, ShcB and ShcC, which are predominantly expressed in neuronal cells, have also been identified. ShcA knockout mice are embryonic lethal and have clearly suggested an important role for ShcA in vivo. Based on dominant negative studies and mouse embryos deficient in ShcA, a clear role for Shc in leading to mitogen activated protein kinase (MAPK) activation has been established. However MAPK activation may not be the sole function of Shc proteins. Although Shc has also been linked to other signaling events such as c-Myc activation and cell survival, the mechanistic understanding of these signaling events remains poorly characterized. Given the apparently central role that Shc plays signaling via many receptors, delineating the precise mechanism(s) of Shc-mediated signaling may be critical to our understanding of the effects mediated through these receptors.

Evidence for a conserved role for CRKII and Rac in engulfment of apoptotic cells.

Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10 are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream of ced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.

Design and use of an inducibly activated human immunodeficiency virus type 1 Nef to study immune modulation.

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the infectivity of virus particles, downmodulate cell surface proteins, and associate with many intracellular proteins that are thought to facilitate HIV infection. One of the challenges in defining the molecular events regulated by Nef has been obtaining good expression of Nef protein in T cells. This has been attributed to effects of Nef on cell proliferation and apoptosis. We have designed a Nef protein that is readily expressed in T-cell lines and whose function is inducibly activated. It is composed of a fusion between full-length Nef and the estrogen receptor hormone-binding domain (Nef-ER). The Nef-ER is kept in an inactive state due to steric hindrance, and addition of the membrane-permeable drug 4-hydroxytamoxifen (4-HT), which binds to the ER domain, leads to inducible activation of Nef-ER within cells. We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains (lipid rafts) only after activation. Using this inducible Nef, we also compared the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T-cell line. Half-maximal downmodulation of cell surface CD4 required very little active Nef-ER and occurred as early as 4 h after addition of 4-HT. In contrast, 50% downmodulation of HLA-A2 by Nef required 16 to 24 h and about 50- to 100-fold-greater concentrations of 4-HT. These data suggest that HLA-A2 downmodulation may require certain threshold levels of active Nef. The differential timing of CD4 and HLA-A2 downmodulation may have implications for HIV pathogenesis and immune evasion.

SHIP inhibits Akt activation in B cells through regulation of Akt membrane localization.

Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co-cross-linking of BCR with the inhibitory receptor Fc gamma RIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to Fc gamma RIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by Fc gamma RIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that Fc gamma RIIB1/SHIP does not inhibit p70(S6k) activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage. (Blood. 2000;96:1449-1456)

p135 src homology 2 domain-containing inositol 5'-phosphatase (SHIPbeta ) isoform can substitute for p145 SHIP in fcgamma RIIB1-mediated inhibitory signaling in B cells.

The inositol 5'-phosphatase, SHIP (also referred to as SHIP-1 or SHIPalpha), is expressed in all cells of the hematopoietic lineage. Depending on the cell type being investigated and the state of differentiation, SHIP isoforms of several different molecular masses (170, 160, 145, 135, 125, and 110 kDa) have been seen in immunoblots. However, the function of the individual isoforms and the effect of expressing multiple isoforms simultaneously are not understood. Some of these SHIP isoforms have recently been characterized at the level of primary sequence. In this report, we investigated the function of the recently characterized 135-kDa SHIP isoform (SHIPbeta), which appears to possess the catalytic domain but lacks some of the protein-protein interaction motifs at the C terminus. By reconstituting SHIP-deficient DT40 B cells with either SHIPbeta or the better-characterized p145 SHIPalpha, we addressed the function of SHIPbeta in the complete absence of SHIPalpha. We observed that SHIPbeta had enzymatic activity comparable with SHIPalpha and that SHIPbeta was able to reconstitute F(c)gammaRIIB1-mediated inhibition of B cell receptor-induced signaling events such as calcium flux and Akt and mitogen-activated protein kinase activation. SHIPbeta was readily phosphorylated in response to B cell receptor cross-linking with the inhibitory receptor F(c)gammaRIIB1 and SHIPbeta also interacted with the adapter protein Shc. During these studies we also observed that the SHIPalpha or SHIPbeta interaction with Grb2 is not required for F(c)gammaRIIB1-mediated inhibition of calcium flux. These data suggest that SHIPbeta, which is normally expressed in B cells along with SHIPalpha, functions comparably with SHIPalpha and that these two isoforms are not likely to be antagonistic in their function in vivo.

Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells.

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling.

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells.

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

A requirement for lipid rafts in B cell receptor induced Ca(2+) flux.

Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio-temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR-induced Ca(2+) flux in B cells. An intact raft structure was required for BCR-induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling.

Neuregulin-increased expression of acetylcholine receptor epsilon-subunit gene requires ErbB interaction with Shc.

Selective transcription of acetylcholine receptor (AChR) subunit genes by neuregulin is one of the mechanisms involved in the synaptic localization of AChRs to the neuromuscular junction. Neuregulin stimulates ErbB receptor tyrosine kinases and subsequently activates the Ras/ERK pathway, which is required for neuregulin-mediated induction of AChR subunit genes in muscle cells and synapse-specific expression in vivo. Here we investigated the neuregulin transduction mechanism that leads to ERK activation after ErbB receptor tyrosine phosphorylation. Neuregulin increases the association of the adaptor proteins Grb2 and Shc with both ErbB2 and ErbB3 in C2C12 muscle cells. Dephosphorylation of the tyrosine-phosphorylated ErbB proteins abolished their association with both Grb2 and Shc, suggesting a tyrosine phosphorylation-dependent interaction. The interaction of Shc with the ErbB receptors is mediated by Shc's phosphotyrosine-binding domain. In addition, neuregulin increased tyrosine phosphorylation of Shc. Mutagenesis approaches demonstrated that tyrosine phosphorylation of Shc is required for neuregulin induction of AChR subunit gene expression. Taken together, these data indicate that the interaction of ErbB receptors with Grb2 alone is insufficient for neuregulin-activated transcription, but that ErbB receptor signaling via Shc is necessary and important.