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The nucleotide oligomerization domain (NOD)-like receptors containing pyrin (NLRP) family of cytosolic pattern-recognition receptors play an integral role in host defense following exposure to a diverse set of pathogenic and sterile threats. The canonical event following ligand recognition is the formation of a heterooligomeric signaling complex termed the inflammasome that produces pro-inflammatory cytokines. Dysregulation of this process is associated with many autoimmune, cardiovascular, metabolic, and neurodegenerative diseases. Despite the range of activating stimuli which affect varied cell types, recent literature makes evident that reactive oxygen species (ROS) are integral to the initiation and propagation of inflammasome signaling. Notably, ROS production and inflammasome activation act in a positive feedback loop to promote this potent immune response. While NLRP3 is by far the most extensively studied NLRP, there is also sufficient literature to make these conclusions for other NLRPs family members. In all cases, a knowledge gap exists regarding the molecular targets and effects of ROS. Future research to define these targets and to parse the order and timing of ROS-mediated NLRP activation will provide meaningful insights into inflammasome biology. This will create novel therapeutic opportunities for the numerous illnesses that are impacted by inflammasome activity.
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Zipper-interacting protein kinase (ZIPK) is a Ser/Thr protein kinase with regulatory involvement in vascular smooth muscle cell (VSMC) actin polymerization and focal adhesion assembly dynamics. ZIPK silencing can induce cytoskeletal remodeling with disassembly of actin stress fiber networks and coincident loss of focal adhesion kinase (FAK)-pY397 phosphorylation. The link between ZIPK inhibition and FAK phosphorylation is unknown, and critical interactor(s) and regulator(s) are not yet defined. In this study, we further analyzed the ZIPK-FAK relationship in VSMCs. The application of HS38, a selective ZIPK inhibitor, to coronary artery vascular smooth muscle cells (CASMCs) suppressed cell migration, myosin light chain phosphorylation (pT18&pS19) and FAK-pY397 phosphorylation as well. This was associated with the translocation of cytoplasmic FAK to the nucleus. ZIPK inhibition with HS38 was consistently found to suppress the activation of FAK and attenuate the phosphorylation of other focal adhesion protein components (i.e., pCas130, paxillin, ERK). In addition, our study showed a decrease in human cell-division cycle 14A phosphatase (CDC14A) levels with ZIPK-siRNA treatment and increased CDC14A with transient transfection of ZIPK. Proximity ligation assays (PLA) revealed CDC14A localized with ZIPK and FAK. Silencing CDC14A showed an increase of FAK-pY397 phosphorylation. Ultimately, the data presented herein strongly support a regulatory mechanism of FAK in CASMCs by a ZIPK-CDC14A partnership; ZIPK may act as a key signal integrator to control CDC14A and FAK during VSMC migration.
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ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-ß-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.
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Neoplasias de la Mama , Inhibidores de Histona Desacetilasas , Femenino , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cromatina , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Vorinostat/farmacologíaRESUMEN
NEW FINDINGS: What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. ABSTRACT: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.
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Arterias Cerebrales , Vasoconstricción , Animales , Humanos , Ratas , Arterias Cerebrales/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Proteínas Quinasas , Resistencia Vascular , Vasoconstricción/fisiologíaRESUMEN
In the field of EEG, researchers generally rely on rules of thumb, rather than a priori statistical calculations, when planning the number of trials to include in an ERP study. To aid in this practice, studies have tried to establish minimum numbers of trials required to reliably isolate ERPs. However, these guidelines do not necessarily apply across different study designs, as the reliability of an ERP waveform is not the same as the statistical power of a given experiment. Experiment parameters such as number of participants, trials, and effect magnitude interact to affect power in complex ways. Both under- and over-powered ERP studies represent a waste of time and resources that impedes the progress of the field. The current study fills this gap by subsampling real ERP data to estimate the relationship between experiment design parameters and statistical power. The simulations include seven commonly studied ERP components: the ERN, LRP, N170, MMN, P3, N2pc, and N400. In the first set of experiments, we determined the probability of obtaining a statistically significant ERP effect for each component. In the second and third set of experiments, we determined the probability of obtaining a statistically significant difference in ERP amplitude within and between groups for each component. Results indicate that the rules of thumb for ERP experiment design in the literature often lead to underpowered studies. Going forward, these results provide researchers with experiment design guidelines that are specific to the component under study, allowing for the design of sufficiently powered ERP studies.
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Electroencefalografía , Potenciales Evocados , Humanos , Masculino , Femenino , Electroencefalografía/métodos , Reproducibilidad de los Resultados , Proyectos de InvestigaciónRESUMEN
The family of death-associated protein kinases (DAPKs) and DAPK-related apoptosis-inducing protein kinases (DRAKs) act as molecular switches for a multitude of cellular processes, including apoptotic and autophagic cell death events. This review summarizes the mechanisms for kinase activity regulation and discusses recent molecular investigations of DAPK and DRAK family members in the intestinal epithelium. In general, recent literature convincingly supports the importance of this family of protein kinases in the homeostatic processes that govern the proper function of the intestinal epithelium. Each of the DAPK family of proteins possesses distinct biochemical properties, and we compare similarities in the information available as well as those cases where functional distinctions are apparent. As the prototypical member of the family, DAPK1 is noteworthy for its tumor suppressor function and association with colorectal cancer. In the intestinal epithelium, DAPK2 is associated with programmed cell death, potential tumor-suppressive functions, and a unique influence on granulocyte biology. The impact of the DRAKs in the epithelium is understudied, but recent studies support a role for DRAK1 in inflammation-mediated tumor growth and metastasis. A commentary is provided on the potential importance of DAPK3 in facilitating epithelial restitution and wound healing during the resolution of colitis. An update on efforts to develop selective pharmacologic effectors of individual DAPK members is also supplied.
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Apoptosis , Neoplasias , Humanos , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Proteínas Reguladoras de la Apoptosis , IntestinosRESUMEN
Smoothelin-like 1 (SMTNL1) modulates the contractile performance of smooth muscle and thus has a key role in vascular homeostasis. Elevated vascular tone, recognized as a contributor to the development of progressive cardiac dysfunction, was previously found with SMTNL1 deletion. In this study, we assessed cardiac morphology and function of male and female, wild-type (Smtnl1+/+) and global SMTNL1 knockout (Smtnl1-/-) mice at 10 weeks of age. Gross dissection revealed distinct cardiac morphology only in males; Smtnl1-/- hearts were significantly smaller than Smtnl1+/+, but the left ventricle (LV) proportion of heart mass was greater. Male Smtnl1-/- mice also displayed increased ejection fraction and fractional shortening, as well as elevated aortic and pulmonary flow velocities. The impact of cardiac stress with pressure overload by transverse aortic constriction (TAC) was examined in male mice. With TAC banding, systolic function was preserved, but the LV filling pressure was selectively elevated due to relaxation impairment. Smtnl1-/- mice displayed higher early/passive filling velocity of LV/early mitral annulus velocity ratio (E/E' ratio) and myocardial performance index along with a prolonged isovolumetric relaxation time. Taken together, the findings support a novel, sex-dimorphic role for SMTNL1 in modulating cardiac structure and function of mice.
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Proteínas Musculares , Músculo Liso , Factores Sexuales , Función Ventricular Izquierda , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Contracción Muscular , Volumen Sistólico , Proteínas Musculares/genética , Fosfoproteínas/genéticaRESUMEN
BACKGROUND: Ulcerative colitis (UC) is a progressive disorder that elevates the risk of colon cancer development through a colitis-dysplasia-carcinoma sequence. Gene expression profiling of colitis-associated lesions obtained from patients with varied extents of UC can be mined to define molecular panels associated with colon cancer development. METHODS: Differential gene expression profiles of 3 UC clinical subtypes and healthy controls were developed for the GSE47908 microarray data set of healthy controls, left-sided colitis, pancolitis, and colitis-associated dysplasia (CAD) using limma R. RESULTS: A gene ontology enrichment analysis of differentially expressed genes (DEGs) revealed a shift in the transcriptome landscape as UC progressed from left-sided colitis to pancolitis to CAD, from being immune-centric to being cytoskeleton-dependent. Hippo signaling (via Yes-associated protein [YAP]) and Ephrin receptor signaling were the top canonical pathways progressively altered in concert with the pathogenic progression of UC. A molecular interaction network analysis of DEGs in left-sided colitis, pancolitis, and CAD revealed 1 pairwise line, or edge, that was topologically important to the network structure. This edge was found to be highly enriched in actin-based processes, and death-associated protein kinase 3 (DAPK3) was a critical member and sole protein kinase member of this network. Death-associated protein kinase 3 is a regulator of actin-cytoskeleton reorganization that controls proliferation and apoptosis. Differential correlation analyses revealed a negative correlation for DAPK3-YAP in healthy controls that flipped to positive in left-sided colitis. With UC progression to CAD, the DAPK3-YAP correlation grew progressively more positive. CONCLUSION: In summary, DAPK3 was identified as a candidate gene involved in UC progression to dysplasia.
Our investigation verified pancolitis as a conduit for ulcerative colitis advancement from left-sided colitis to dysplasia and uniquely identified dysregulation of actin reorganization, with death-associated protein kinase 3 and Yes-associated protein as key molecular determinants for disease progression.
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Colitis Ulcerosa , Colitis , Neoplasias del Colon , Actinas/metabolismo , Colitis/complicaciones , Colitis Ulcerosa/complicaciones , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/complicaciones , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Efrinas/metabolismo , Humanos , Hiperplasia/complicaciones , Proteínas Señalizadoras YAPRESUMEN
Insulin resistance (InR) is manifested in skeletal muscle by decreased insulin-stimulated glucose uptake due to impaired insulin signaling and multiple post-receptor intracellular defects. Chronic glucose-induced insulin resistance leads to the activation of Ser/Thr kinases and elevated phosphorylation of insulin receptor substrate 1 (IRS1) on Ser residues. Phosphorylation of IRS1 triggers the dissociation of IRS1 and its downstream effector, phosphatidylinositol 3-kinase. In the present study, we provide evidence for the insulin-sensitizing role of smoothelin-like protein 1 (SMTNL1) that is a ligand-dependent co-regulator of steroid receptors, predominantly the progesterone receptor. SMTNL1 was transiently overexpressed in insulin-resistant C2C12 myotubes. A proteome profiler array revealed that mTOR and Ser/Thr kinases were SMTNL1-dependent signaling pathways. In the presence of progesterone, overexpression was coupled to decreased Ser phosphorylation of IRS1 at Ser307, Ser318, and Ser612 residues. SMTNL1 also induced the expression and activity of the p85 subunit of PI3K. SMTNL1 regulated the expression of PKCε, which phosphorylates IRS1 at Ser318 residue. SMTNL1 also regulated ERK1/2 and JNK, which phosphorylate IRS1 at Ser612 and Ser307, respectively. Real-time metabolic measurements of oxygen consumption rate and extracellular acidification rate revealed that SMTNL1 improved glycolysis and promoted the utilization of alternative carbon fuels. SMTNL1 also rescued the mitochondrial respiration defect induced by chronic insulin exposure. Collectively, SMTNL1 plays a crucial role in maintaining the physiological ratio of Tyr/Ser IRS1 phosphorylation and attenuates the insulin-signaling cascade that contributes to impaired glucose disposal, which makes it a potential therapeutic target for improving InR.
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Resistencia a la Insulina , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Animales , Glucosa/metabolismo , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , FosforilaciónRESUMEN
Advances in high-throughput sequencing technologies now yield unprecedented volumes of OMICs data with opportunities to conduct systematic data analyses and derive novel biological insights. Here, we provide protocols to perform differential-expressed gene analysis of TCGA and GTEx RNA-Seq data from human cancers, complete integrative GO and network analyses with focus on clinical and survival data, and identify differential correlation of trait-associated biomarkers. For complete details on the use and execution of this protocol, please refer to Chen and MacDonald (2021).
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Neoplasias , Biomarcadores de Tumor/genética , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias/diagnóstico , RNA-SeqRESUMEN
Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 µM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; Kd1: 3.6 µM, Kd2: 113.6 µM, respectively). A recombinant AQP4 protein domain (recAQP4CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 µM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data support a unique adapter protein binding mode for CaM with AQP4.
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Acuaporina 4/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Secuencia de Aminoácidos , Acuaporina 4/química , Sitios de Unión , Calmodulina/química , Humanos , Unión Proteica , Conformación Proteica , Dominios ProteicosRESUMEN
Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.
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Catepsina Z , Inflamasomas , Animales , Catepsina Z/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Integrina alfa5/metabolismo , Interleucina-1beta/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Dióxido de Silicio/farmacología , Silicosis/metabolismoRESUMEN
Colon adenocarcinoma is a prevalent malignancy with significant mortality. Hence, the identification of molecular biomarkers with prognostic significance is important for improved treatment and patient outcomes. Clinical traits and RNA-Seq of 551 patient samples in the UCSC Toil Recompute Compendium of The Cancer Genome Atlas TARGET and Genotype Tissue Expression project datasets (primary_site = colon) were used for weighted gene co-expression network analysis to reveal the association between gene networks and cancer cell invasion. One module, containing 151 genes, was significantly correlated with lymphatic invasion, a histopathological feature of higher risk colon cancer. DAPK3 (death-associated protein kinase 3) was identified as the pseudohub of the module. Gene ontology identified gene enrichment related to cytoskeletal organization and apoptotic signaling processes, suggesting modular involvement in tumor cell survival, migration, and epithelial-mesenchymal transformation. Although DAPK3 expression was reduced in patients with colon cancer, high expression of DAPK3 was significantly correlated with greater lymphatic invasion and poor overall survival.
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In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1ß release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1ß release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein-ligand binding that might explain the activity of the compounds.
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Imidazoles , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Humanos , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/farmacología , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células THP-1RESUMEN
OBJECTIVE: The effectiveness of three types of in-vehicle warnings was assessed in a driving simulator across different noise conditions. BACKGROUND: Although there has been much research comparing different types of warnings in auditory displays and interfaces, many of these investigations have been conducted in quiet laboratory environments with little to no consideration of background noise. Furthermore, the suitability of some auditory warning types, such as spearcons, as car warnings has not been investigated. METHOD: Two experiments were conducted to assess the effectiveness of three auditory warnings (spearcons, text-to-speech, auditory icons) with different types of background noise while participants performed a simulated driving task. RESULTS: Our results showed that both the nature of the background noise and the type of auditory warning influenced warning recognition accuracy and reaction time. Spearcons outperformed text-to-speech warnings in relatively quiet environments, such as in the baseline noise condition where no music or talk-radio was played. However, spearcons were not better than text-to-speech warnings with other background noises. Similarly, the effectiveness of auditory icons as warnings fluctuated across background noise, but, overall, auditory icons were the least efficient of the three warning types. CONCLUSION: Our results supported that background noise can have an idiosyncratic effect on a warning's effectiveness and illuminated the need for future research into ameliorating the effects of background noise. APPLICATION: This research can be applied to better present warnings based on the anticipated auditory environment in which they will be communicated.
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Conducción de Automóvil , Música , Humanos , Tiempo de ReacciónRESUMEN
The pryin domain (PYD) domain is involved in protein interactions that lead to assembly of immune-sensing complexes such as inflammasomes. The repertoire of PYD-containing genes expressed by a cell type arms tissues with responses against a range of stimuli. The transcriptional regulation of the PYD gene family however is incompletely understood. Alternative promoter utilization was identified as a mechanism regulating the tissue distribution of human PYD gene family members, including NLRP6 that is translationally silenced outside of intestinal tissue. Results show that alternative transcriptional promoters mediate NLRP6 silencing in mice and humans, despite no upstream genomic synteny. Human NLRP6 contains an internal alternative promoter within exon 2 of the PYD, resulting in a truncated mRNA in nonintestinal tissue. In mice, a proximal promoter was used that expanded the 5' leader sequence restricting nuclear export and abolishing translational efficiency. Nlrp6 was dispensable in disease models targeting the kidney, which expresses noncanonical isoforms. Thus, alternative promoter use is a critical mechanism not just for isoform modulation but for determining expression profile and function of PYD family members.
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Empalme Alternativo/genética , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Corteza Renal/metabolismo , Regiones Promotoras Genéticas/genética , Dominio Pirina/genética , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Animales , Células Cultivadas , Exones , Expresión Génica , Regulación de la Expresión Génica , Genes Reguladores , Humanos , Inflamasomas/metabolismo , Mucosa Intestinal/patología , Corteza Renal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismoRESUMEN
The prototypical model for NOD-like receptor (NLR) inflammasome assembly includes nucleotide-dependent activation of the NLR downstream of pathogen- or danger-associated molecular pattern (PAMP or DAMP) recognition, followed by nucleation of hetero-oligomeric platforms that lie upstream of inflammatory responses associated with innate immunity. As members of the STAND ATPases, the NLRs are generally thought to share a similar model of ATP-dependent activation and effect. However, recent observations have challenged this paradigm to reveal novel and complex biochemical processes to discern NLRs from other STAND proteins. In this review, we highlight past findings that identify the regulatory importance of conserved ATP-binding and hydrolysis motifs within the nucleotide-binding NACHT domain of NLRs and explore recent breakthroughs that generate connections between NLR protein structure and function. Indeed, newly deposited NLR structures for NLRC4 and NLRP3 have provided unique perspectives on the ATP-dependency of inflammasome activation. Novel molecular dynamic simulations of NLRP3 examined the active site of ADP- and ATP-bound models. The findings support distinctions in nucleotide-binding domain topology with occupancy of ATP or ADP that are in turn disseminated on to the global protein structure. Ultimately, studies continue to reveal how the ATP-binding and hydrolysis properties of NACHT domains in different NLRs integrate with signaling modules and binding partners to control innate immune responses at the molecular level.
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Adenosina Trifosfato/metabolismo , Inflamasomas/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Humanos , Hidrólisis , Simulación de Dinámica Molecular , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.
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Acuaporina 4/metabolismo , Edema/metabolismo , Edema/terapia , Animales , Acuaporina 4/fisiología , Astrocitos/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Calmodulina/metabolismo , Sistema Nervioso Central/metabolismo , Edema/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Trifluoperazina/farmacologíaRESUMEN
Speech comprehension is often thought of as an entirely auditory process, but both normal hearing and hearing-impaired individuals sometimes use visual attention to disambiguate speech, particularly when it is difficult to hear. Many studies have investigated how visual attention (or the lack thereof) impacts the perception of simple speech sounds such as isolated consonants, but there is a gap in the literature concerning visual attention during natural speech comprehension. This issue needs to be addressed, as individuals process sounds and words in everyday speech differently than when they are separated into individual elements with no competing sound sources or noise. Moreover, further research is needed to explore patterns of eye movements during speech comprehension - especially in the presence of noise - as such an investigation would allow us to better understand how people strategically use visual information while processing speech. To this end, we conducted an experiment to track eye-gaze behavior during a series of listening tasks as a function of the number of speakers, background noise intensity, and the presence or absence of simulated hearing impairment. Our specific aims were to discover how individuals might adapt their oculomotor behavior to compensate for the difficulty of the listening scenario, such as when listening in noisy environments or experiencing simulated hearing loss. Speech comprehension difficulty was manipulated by simulating hearing loss and varying background noise intensity. Results showed that eye movements were affected by the number of speakers, simulated hearing impairment, and the presence of noise. Further, findings showed that differing levels of signal-to-noise ratio (SNR) led to changes in eye-gaze behavior. Most notably, we found that the addition of visual information (i.e. videos vs. auditory information only) led to enhanced speech comprehension - highlighting the strategic usage of visual information during this process.
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Incomplete block designs are experimental designs in which a subset of treatments are included in each block. The researcher must decide which conditions are assigned to each block. This design concept is quite general. At the level of the experiment, a treatment is a condition in an experiment, blocks are different groups of subjects, and the researcher must decide how to assign a subset of conditions to each block of subjects. At the level of the subject, the treatments correspond to individual stimuli, blocks correspond to experimental trials, and the researcher must decide which subset of stimuli to include in each trial. In this article, we present an efficient algorithm that assigns treatments to blocks in an incomplete block design according to two criteria: Each pair of treatments must appear together in at least one block, and the number of blocks in the experiment is minimized. We discuss details and applications of the algorithm and provide software and a web application to generate designs according to the needs of the researcher.