The adaptor molecule ADAP and its paralog, PRAM-1, are expressed in mononuclear cells, lymphocyte... more The adaptor molecule ADAP and its paralog, PRAM-1, are expressed in mononuclear cells, lymphocytes, and granulocytes. ADAP plays a crucial role in T cell receptor-induced integrin activation, conjugate formation, NF-kB activation, IL-2 production, and T cell proliferation. However, it is not clear how ADAP exerts its profound effects on T cell activation. Here, we demonstrate that endogenous ADAP is an integral component of TCR-induced SLP-76 microclusters, and is essential for their persistence and movement. Our live cell imaging studies show that when we break the ADAP-SLP-76 interaction ADAP is not recruited into SLP-76 MC and SLP-76 MC are immobile and non-persistent. Consequently, T cell adhesion via the TCR is severely impaired, indicating a novel role for ADAP in adhesion to TCR ligands. Phosphorylation of highly conserved, SLP-76-interacting tyrosines within the ADAP C-terminus is necessary for ADAP to enter SLP-76 MC, and associations mediated by the N-terminus are required...
The adapter molecule SKAP55 enables the formation of T-cell:APC conjugates, facilitates T-cell cy... more The adapter molecule SKAP55 enables the formation of T-cell:APC conjugates, facilitates T-cell cytokine production, and is required for optimal T cell proliferation in response to antigen. However, the molecular basis for the involvement of SKAP55 in these events remains unclear. Here, we demonstrate that both endogenous SKAP55 and exogenous SKAP55 chimeras enter T cell receptor (TCR)-induced signaling microclusters containing the adapter protein SLP-76. Using a SKAP55-deficient Jurkat T cell line, we demonstrate that SKAP55 is required for both the persistence and the movement of SLP-76 microclusters. Furthermore, the recruitment of SKAP55 into microclusters requires the presence of both SLP-76 and ADAP, an intermediary adapter protein capable of interacting with both SLP-76 and SKAP55. In addition, we show that SKAP55 homodimerizes via an N-terminal dimerization motif (DM). Our structure-function analyses indicate that the dimerization domain and the ADAP-interacting Src-homology ...
International Journal of Business & Technology, 2015
Captcha (Completely Automated Public Turing test to tell Computers and Humans Apart) is a widely ... more Captcha (Completely Automated Public Turing test to tell Computers and Humans Apart) is a widely used online security tool that ensures that a computer program is not posing as a human user. While smart programs with advanced image processing capability have already cracked picture based captcha systems there is a need for making the test harder. This paper presents a design prototype of a simplified type of labeled-image captcha where a picture of a common animal or household item is marked with a number of different labels and the users will be asked to provide the correct label for specific parts of the picture. Due to human’s familiarity with body shapes and part names of such common pictures, they will easily identify a specific organ/parts of the picture. Such labeled-image captcha tests are expected to be very easy for human users regardless of their culture, age, gender, educational background and other discriminations but tough for the bots and automated computer programs.
Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitabili... more Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitability, thus supporting many complex behaviors. Perturbed synaptic and extrasynaptic Glu homeostasis in the human brain has been implicated in multiple neuropsychiatric and neurodegenerative disorders including Parkinson's disease, where theories suggest that excitotoxic insults may accelerate a naturally occurring process of dopamine (DA) neuron degeneration. In C. elegans, mutation of the glial expressed gene, swip-10, results in Glu-dependent DA neuron hyperexcitation that leads to elevated DA release, triggering DA signaling-dependent motor paralysis. Here, we demonstrate that swip-10 mutations induce premature and progressive DA neuron degeneration, with light and electron microscopy studies demonstrating the presence of dystrophic dendritic processes, as well as shrunken and/or missing cell soma. As with paralysis, DA neuron degeneration in swip-10 mutants is rescued by glial-speci...
Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs)... more Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases,...
We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle atta... more We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in C. elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane (UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin) and UNC-95), are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adj...
Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and r... more Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated h...
RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variet... more RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variety of mechanisms to ensure the proper post-transcriptional regulation of their target RNAs. RBPs in multiple species have been identified as playing crucial roles during development and as having important functions in various adult organ systems, including the heart, nervous, muscle, and reproductive systems. ETR-1, a highly conserved ELAV-Type RNA-binding protein belonging to the CELF/Bruno protein family, has been previously reported to be involved in C. elegans muscle development. Animals depleted of ETR-1 have been previously characterized as arresting at the two-fold stage of embryogenesis. In this study, we show that ETR-1 is expressed in the hermaphrodite somatic gonad and germ line, and that reduction of ETR-1 via RNA interference (RNAi) results in reduced hermaphrodite fecundity. Detailed characterization of this fertility defect indicates that ETR-1 is required in both the soma...
Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range... more Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range of pathologies called ciliopathies. Ciliary specialization in form and function is observed throughout the animal kingdom, yet mechanisms generating ciliary diversity are poorly understood. The "tubulin code"-a combination of tubulin isotypes and tubulin post-translational modifications-can generate microtubule diversity. Using C. elegans, we show that α-tubulin isotype TBA-6 sculpts 18 A- and B-tubule singlets from nine ciliary A-B doublet microtubules in cephalic male (CEM) neurons. In CEM cilia, tba-6 regulates velocities and cargoes of intraflagellar transport (IFT) kinesin-2 motors kinesin-II and OSM-3/KIF17 without affecting kinesin-3 KLP-6 motility. In addition to their unique ultrastructure and accessory kinesin-3 motor, CEM cilia are specialized to produce extracellular vesicles. tba-6 also influences several aspects of extracellular vesicle biology, including cargo s...
The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote... more The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 μm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neuro...
The differentiated cell identities and structure of fully formed organs are generally stable afte... more The differentiated cell identities and structure of fully formed organs are generally stable after their development. In contrast, we report here that development of the C. elegans proximal somatic gonad (hermaphrodite uterus and spermathecae, and male vas deferens) can be redirected into intestine-like organs by brief expression of the ELT-7 GATA transcription factor. This process converts one developing organ into another and can hence be considered "transorganogenesis." We show that, following pulsed ELT-7 expression, cells of the uterus activate and maintain intestine-specific gene expression and are transformed at the ultrastructural level to form an epithelial tube resembling the normal intestine formed during embryogenesis. Ubiquitous ELT-7 expression activates intestinal markers in many different cell types but only cells in the somatic gonad and pharynx appear to become fully reprogrammed. We found that ectopic expression of other endoderm-promoting transcription ...
Syndapin/Pascin family F-BAR domain proteins bind directly to membrane lipids and are associated ... more Syndapin/Pascin family F-BAR domain proteins bind directly to membrane lipids and are associated with actin dynamics at the plasma membrane. Previous reports have also implicated mammalian syndapin 2 in endosome function during receptor recycling, but precise analysis of a putative recycling function for syndapin in mammalian systems is difficult because of syndapin effects on the earlier step of endocytic uptake, and potential redundancy among the three separate genes that encode mammalian syndapin isoforms. Here we analyze the endocytic transport function of the only C. elegans syndapin, SDPN-1. We find that SDPN-1 is a resident protein of the early and basolateral recycling endosomes in the C. elegans intestinal epithelium, and sdpn-1 deletion mutants display phenotypes indicating a block in basolateral recycling transport. sdpn-1 mutants accumulate abnormal endosomes positive for early endosome and recycling endosome markers that are normally separate, and such endosomes accumul...
Proceedings of the National Academy of Sciences of the United States of America, Jul 11, 2016
Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor... more Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in hum...
The adaptor molecule ADAP and its paralog, PRAM-1, are expressed in mononuclear cells, lymphocyte... more The adaptor molecule ADAP and its paralog, PRAM-1, are expressed in mononuclear cells, lymphocytes, and granulocytes. ADAP plays a crucial role in T cell receptor-induced integrin activation, conjugate formation, NF-kB activation, IL-2 production, and T cell proliferation. However, it is not clear how ADAP exerts its profound effects on T cell activation. Here, we demonstrate that endogenous ADAP is an integral component of TCR-induced SLP-76 microclusters, and is essential for their persistence and movement. Our live cell imaging studies show that when we break the ADAP-SLP-76 interaction ADAP is not recruited into SLP-76 MC and SLP-76 MC are immobile and non-persistent. Consequently, T cell adhesion via the TCR is severely impaired, indicating a novel role for ADAP in adhesion to TCR ligands. Phosphorylation of highly conserved, SLP-76-interacting tyrosines within the ADAP C-terminus is necessary for ADAP to enter SLP-76 MC, and associations mediated by the N-terminus are required...
The adapter molecule SKAP55 enables the formation of T-cell:APC conjugates, facilitates T-cell cy... more The adapter molecule SKAP55 enables the formation of T-cell:APC conjugates, facilitates T-cell cytokine production, and is required for optimal T cell proliferation in response to antigen. However, the molecular basis for the involvement of SKAP55 in these events remains unclear. Here, we demonstrate that both endogenous SKAP55 and exogenous SKAP55 chimeras enter T cell receptor (TCR)-induced signaling microclusters containing the adapter protein SLP-76. Using a SKAP55-deficient Jurkat T cell line, we demonstrate that SKAP55 is required for both the persistence and the movement of SLP-76 microclusters. Furthermore, the recruitment of SKAP55 into microclusters requires the presence of both SLP-76 and ADAP, an intermediary adapter protein capable of interacting with both SLP-76 and SKAP55. In addition, we show that SKAP55 homodimerizes via an N-terminal dimerization motif (DM). Our structure-function analyses indicate that the dimerization domain and the ADAP-interacting Src-homology ...
International Journal of Business & Technology, 2015
Captcha (Completely Automated Public Turing test to tell Computers and Humans Apart) is a widely ... more Captcha (Completely Automated Public Turing test to tell Computers and Humans Apart) is a widely used online security tool that ensures that a computer program is not posing as a human user. While smart programs with advanced image processing capability have already cracked picture based captcha systems there is a need for making the test harder. This paper presents a design prototype of a simplified type of labeled-image captcha where a picture of a common animal or household item is marked with a number of different labels and the users will be asked to provide the correct label for specific parts of the picture. Due to human’s familiarity with body shapes and part names of such common pictures, they will easily identify a specific organ/parts of the picture. Such labeled-image captcha tests are expected to be very easy for human users regardless of their culture, age, gender, educational background and other discriminations but tough for the bots and automated computer programs.
Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitabili... more Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitability, thus supporting many complex behaviors. Perturbed synaptic and extrasynaptic Glu homeostasis in the human brain has been implicated in multiple neuropsychiatric and neurodegenerative disorders including Parkinson's disease, where theories suggest that excitotoxic insults may accelerate a naturally occurring process of dopamine (DA) neuron degeneration. In C. elegans, mutation of the glial expressed gene, swip-10, results in Glu-dependent DA neuron hyperexcitation that leads to elevated DA release, triggering DA signaling-dependent motor paralysis. Here, we demonstrate that swip-10 mutations induce premature and progressive DA neuron degeneration, with light and electron microscopy studies demonstrating the presence of dystrophic dendritic processes, as well as shrunken and/or missing cell soma. As with paralysis, DA neuron degeneration in swip-10 mutants is rescued by glial-speci...
Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs)... more Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases,...
We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle atta... more We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in C. elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane (UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin) and UNC-95), are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adj...
Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and r... more Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated h...
RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variet... more RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variety of mechanisms to ensure the proper post-transcriptional regulation of their target RNAs. RBPs in multiple species have been identified as playing crucial roles during development and as having important functions in various adult organ systems, including the heart, nervous, muscle, and reproductive systems. ETR-1, a highly conserved ELAV-Type RNA-binding protein belonging to the CELF/Bruno protein family, has been previously reported to be involved in C. elegans muscle development. Animals depleted of ETR-1 have been previously characterized as arresting at the two-fold stage of embryogenesis. In this study, we show that ETR-1 is expressed in the hermaphrodite somatic gonad and germ line, and that reduction of ETR-1 via RNA interference (RNAi) results in reduced hermaphrodite fecundity. Detailed characterization of this fertility defect indicates that ETR-1 is required in both the soma...
Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range... more Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range of pathologies called ciliopathies. Ciliary specialization in form and function is observed throughout the animal kingdom, yet mechanisms generating ciliary diversity are poorly understood. The "tubulin code"-a combination of tubulin isotypes and tubulin post-translational modifications-can generate microtubule diversity. Using C. elegans, we show that α-tubulin isotype TBA-6 sculpts 18 A- and B-tubule singlets from nine ciliary A-B doublet microtubules in cephalic male (CEM) neurons. In CEM cilia, tba-6 regulates velocities and cargoes of intraflagellar transport (IFT) kinesin-2 motors kinesin-II and OSM-3/KIF17 without affecting kinesin-3 KLP-6 motility. In addition to their unique ultrastructure and accessory kinesin-3 motor, CEM cilia are specialized to produce extracellular vesicles. tba-6 also influences several aspects of extracellular vesicle biology, including cargo s...
The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote... more The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 μm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neuro...
The differentiated cell identities and structure of fully formed organs are generally stable afte... more The differentiated cell identities and structure of fully formed organs are generally stable after their development. In contrast, we report here that development of the C. elegans proximal somatic gonad (hermaphrodite uterus and spermathecae, and male vas deferens) can be redirected into intestine-like organs by brief expression of the ELT-7 GATA transcription factor. This process converts one developing organ into another and can hence be considered "transorganogenesis." We show that, following pulsed ELT-7 expression, cells of the uterus activate and maintain intestine-specific gene expression and are transformed at the ultrastructural level to form an epithelial tube resembling the normal intestine formed during embryogenesis. Ubiquitous ELT-7 expression activates intestinal markers in many different cell types but only cells in the somatic gonad and pharynx appear to become fully reprogrammed. We found that ectopic expression of other endoderm-promoting transcription ...
Syndapin/Pascin family F-BAR domain proteins bind directly to membrane lipids and are associated ... more Syndapin/Pascin family F-BAR domain proteins bind directly to membrane lipids and are associated with actin dynamics at the plasma membrane. Previous reports have also implicated mammalian syndapin 2 in endosome function during receptor recycling, but precise analysis of a putative recycling function for syndapin in mammalian systems is difficult because of syndapin effects on the earlier step of endocytic uptake, and potential redundancy among the three separate genes that encode mammalian syndapin isoforms. Here we analyze the endocytic transport function of the only C. elegans syndapin, SDPN-1. We find that SDPN-1 is a resident protein of the early and basolateral recycling endosomes in the C. elegans intestinal epithelium, and sdpn-1 deletion mutants display phenotypes indicating a block in basolateral recycling transport. sdpn-1 mutants accumulate abnormal endosomes positive for early endosome and recycling endosome markers that are normally separate, and such endosomes accumul...
Proceedings of the National Academy of Sciences of the United States of America, Jul 11, 2016
Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor... more Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in hum...
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Papers by Ken Nguyễn