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Silicosis due to artificial stone (AS) has emerged over the last decade as an increasing global issue. We report the first eight UK cases. All were men; median age was 34 years (range 27-56) and median stone dust exposure was 12.5 years (range 4-40) but in 4 cases was 4-8 years. One is deceased; two were referred for lung transplant assessment. All cases were dry cutting and polishing AS worktops with inadequate safety measures. Clinical features of silicosis can closely mimic sarcoidosis. UK cases are likely to increase, with urgent action needed to identify cases and enforce regulations.
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Inclusion of a phase change material such as germanium-antimony-telluride (Ge2Sb2Te5 or GST) enhances the performance of plasmonic metasurface absorbers (PMAs). One-dimensional (1-D) plasmonic metasurfaces (PMs) support the excitation of surface plasmon modes for the normal incidence of transverse magnetically (TM) polarized light. The 1-D PMAs absorb incident light because of their confinement in the groove region, which is possible because of the surface plasmon modes excited at the metal-dielectric interface. A thin layer of the phase change material enhances the absorption of incident light because of the increasing strength of the confined electromagnetic field in the vicinity of the PMA. We developed a GST loaded, low cost, 1-D PMA for the absorption of near-infrared (NIR) light (740-920â nm). The PMA was fabricated using an Ag coated 1-D patterned polycarbonate, which was obtained from a commercial digital versatile disk (DVD). A 1-D PMA of 1â cm2 in size obtained from a DVD was coated with a GST layer of 8â nm in thickness to achieve the maximum absorption of 99.56% for the hexagonal closed packed (h.c.p.) crystalline state of the GST loaded layer. Control experiments were performed for different temperatures and different thicknesses of the GST layer for achieving an optimal performance nearing perfect absorption. Electric and magnetic field profiles were simulated for the normal incidence of TM-polarized light to understand the underlying physics of the light-matter interaction with the PMA. Such a PMA can be used to develop various cost-effective optical devices, such as optical sensors, optical filters, photodetectors, and heat absorbing photonic windows in the NIR wavelength regime.
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Background: Resilience is the capacity to bounce back from adversity. Severe mental illnesses are associated with poor and heterogeneous functional outcomes. Symptom remission is inadequate to achieve patient-oriented outcome, and positive psychopathology constructs like resilience have emerged as possible mediators. An exploration of resilience and its association with functional outcomes can drive therapeutic endeavors. Aim: To assess and compare the influence of resilience on disability among patients diagnosed and treated for bipolar disorder and schizophrenia in a tertiary care facility. Methods: Study design - Hospital-based, cross-sectional, comparative design; study population - patients of bipolar disorder and schizophrenia with 2-5 years illness and Clinical Global Impression - Severity (CGI-S) <4; sampling procedure - consecutive sampling; sample size - 30 patients each; scales used - Connor-Davidson Resilience Scale (CD-RISC), Indian Disability Evaluation and Assessment Scale (IDEAS), and CGI-S; patients were evaluated with IDEAS, and 15 persons with and without a significant disability were recruited in each group of schizophrenia and bipolar disorder. Results: The mean CD-RISC 25 score for persons with schizophrenia was 73.60 ± 13.87, whereas that for persons with bipolar disorder was 78.10 ± 15.26. For schizophrenia, only CDRISC-25 scores are statistically significant (t = -2.582, P = 0.018) for predicting IDEAS global disability. For bipolar disorder, CDRISC-25 scores (t = -2.977, P = 0.008) and CGI-severity scores (t = 3.135, P = 0.005) are statistically significant for predicting IDEAS global disability. Conclusion: When disability is factored in, resilience is comparable in persons with schizophrenia and bipolar disorder. Resilience independently predicts disability in both groups. However, the type of disorder does not significantly affect the relationship between resilience and disability. Irrespective of diagnosis, higher resilience is associated with lower disability.
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Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressin-regulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/ and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressin-induced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies.NEW & NOTEWORTHY Vasopressin regulates renal water excretion through control of the aquaporin-2 water channel in collecting duct cells. Studies of vasopressin-induced protein phosphorylation have focused mainly on the phosphorylation of aquaporin-2. This study describes 44 phosphoproteins other than aquaporin-2 that undergo vasopressin-mediated phosphorylation changes and summarizes potential physiological roles of each.
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Aquaporina 2 , Túbulos Renais Coletores , Ratos , Camundongos , Animais , Aquaporina 2/metabolismo , Túbulos Renais Coletores/metabolismo , Fosforilação , Vasopressinas/farmacologia , Vasopressinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfoproteínas/metabolismo , Água/metabolismoRESUMO
The advent of modern quantitative protein mass spectrometry techniques around the turn of the 21st century has contributed to a revolution in biology referred to as 'systems biology'. These methods allow identification and quantification of thousands of proteins in a biological specimen, as well as detection and quantification of post-translational protein modifications including phosphorylation. Here, we discuss these methodologies and show how they can be applied to understand the effects of the peptide hormone vasopressin to regulate the molecular water channel aquaporin-2. The emerging picture provides a detailed framework for understanding the molecular mechanisms involved in water balance disorders.
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Lacunar infarction (LACI), a subtype of acute ischemic stroke, has poor mid- to long-term prognosis due to recurrent vascular events or incident dementia which is difficult to predict using existing clinical data. Herein, we aim to discover blood-based biomarkers for LACI as a complementary prognostic tool. Convalescent plasma was collected from forty-five patients following a non-disabling LACI along with seventeen matched control subjects. The patients were followed up prospectively for up to five years to record an occurrence of adverse outcome and grouped accordingly (i.e., LACI-no adverse outcome, LACI-recurrent vascular event, and LACI-cognitive decline without any recurrence of vascular events). Medium-sized extracellular vesicles (MEVs), isolated from the pooled plasma of four groups, were analyzed by stable isotope labeling and 2D-LC-MS/MS. Out of 573 (FDR < 1%) quantified proteins, 146 showed significant changes in at least one LACI group when compared to matched healthy control. A systems analysis revealed that major elements (~85%) of the MEV proteome are different from the proteome of small-sized extracellular vesicles obtained from the same pooled plasma. The altered MEV proteins in LACI patients are mostly reduced in abundance. The majority of the shortlisted MEV proteins are not linked to commonly studied biological processes such as coagulation, fibrinolysis, or inflammation. Instead, they are linked to oxygen-glucose deprivation, endo-lysosomal trafficking, glucose transport, and iron homeostasis. The dataset is provided as a web-based data resource to facilitate meta-analysis, data integration, and targeted large-scale validation.
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Vesículas Extracelulares , AVC Isquêmico , Acidente Vascular Cerebral Lacunar , Biomarcadores/metabolismo , Cromatografia Líquida , Vesículas Extracelulares/metabolismo , Glucose , Humanos , Ferro , Oxigênio , Prognóstico , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em TandemRESUMO
Intracellular protein gradients serve a variety of functions, such as the establishment of cell polarity or to provide positional information for gene expression in developing embryos. Given that cell size in a population can vary considerably, for the protein gradients to work properly they often have to be scaled to the size of the cell. Here, we examine a model of protein gradient formation within a cell that relies on cytoplasmic diffusion and cortical transport of proteins toward a cell pole. We show that the shape of the protein gradient is determined solely by the cell geometry. Furthermore, we show that the length scale over which the protein concentration in the gradient varies is determined by the linear dimensions of the cell, independent of the diffusion constant or the transport speed. This gradient provides scale-invariant positional information within a cell, which can be used for assembly of intracellular structures whose size is scaled to the linear dimensions of the cell, such as the cytokinetic ring and actin cables in budding yeast cells.
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Actinas , Saccharomycetales , Actinas/metabolismo , Polaridade Celular , Citoplasma/metabolismo , Difusão , Saccharomycetales/metabolismoRESUMO
Oxygen glucose deprivation (OGD) of brain cells is the commonest in vitro model of ischemic stroke that is used extensively for basic and preclinical stroke research. Protein mass spectrometry is one of the most promising and rapidly evolving technologies in biomedical research. A systems-level understanding of cell-type-specific responses to oxygen and glucose deprivation without systemic influence is a prerequisite to delineate the response of the neurovascular unit following ischemic stroke. In this systematic review, we summarize the proteomics studies done on different OGD models. These studies have followed an expression or interaction proteomics approach. They have been primarily used to understand the cellular pathophysiology of ischemia-reperfusion injury or to assess the efficacy of interventions as potential treatment options. We compile the limitations of OGD model and downstream proteomics experiment. We further show that despite having limitations, several proteins shortlisted as altered in in vitro OGD-proteomics studies showed comparable regulation in ischemic stroke patients. This showcases the translational potential of this approach for therapeutic target and biomarker discovery. We next discuss the approaches that can be adopted for cell-type-specific validation of OGD-proteomics results in the future. Finally, we briefly present the research questions that can be addressed by OGD-proteomics studies using emerging techniques of protein mass spectrometry. We have also created a web resource compiling information from OGD-proteomics studies to facilitate data sharing for community usage. This review intends to encourage preclinical stroke community to adopt a hypothesis-free proteomics approach to understand cell-type-specific responses following ischemic stroke.
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Isquemia Encefálica , AVC Isquêmico , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Isquemia Encefálica/metabolismo , Glucose/metabolismo , Humanos , Oxigênio/metabolismo , Proteômica , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
Systems biology can be defined as the study of a biological process in which all of the relevant components are investigated together in parallel to discover the mechanism. Although the approach is not new, it has come to the forefront as a result of genome sequencing projects completed in the first few years of the current century. It has elements of large-scale data acquisition (chiefly next-generation sequencing-based methods and protein mass spectrometry) and large-scale data analysis (big data integration and Bayesian modeling). Here we discuss these methodologies and show how they can be applied to understand the downstream effects of GPCR signaling, specifically looking at how the neurohypophyseal peptide hormone vasopressin, working through the V2 receptor and PKA activation, regulates the water channel aquaporin-2. The emerging picture provides a detailedframework for understanding the molecular mechanisms involved in water balance disorders, pointing the way to improved treatment of both polyuric disorders and water-retention disorders causing dilutional hyponatremia.
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Receptores de Vasopressinas , Desequilíbrio Hidroeletrolítico , Aquaporina 2/metabolismo , Teorema de Bayes , Humanos , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Biologia de SistemasRESUMO
In this article, for the first time, we explained a detailed physical insight for negative differential resistance (NDR) to positive differential resistance (PDR) transition in a ferroelectric (FE)-based negative capacitance (NC) FET and also its dependence on the device terminal voltages. Using extensive well-calibrated TCAD simulations, we have investigated this phenomenon on fully depleted silicon on insulator (FDSOI)-NCFET. The NDR-to-PDR transition occurs due to FE layer capacitance changes from a negative to positive state during channel pinchoff. This, in turn, results in a valley point in the output characteristic ( IDS - VDS ) at which the output resistance is infinite. We also found that we could alter the valley point location by modulating the vertical electric field through the FE layer in the channel pinchoff region using body bias ( VBB ). The interface oxide charges also impacted the NDR to PDR transition, and a positive interface charge results in faster NDR to PDR transition. Furthermore, we have utilized the modulation in the NDR-to-PDR transition due to VBB for designing a current mirror. Results show that the output current ( IOUT ) variation due to VDS reduces from ~8% to ~2% with VBB . We have also designed a single-stage common source (CS) amplifier and provided design guidelines to achieve a higher gain in the NDR region. The results obtained using a small-signal model of the FDSOI-NCFET demonstrate that ~25% higher gain can be achieved with the discussed design guidelines in the NDR region compared to the transition region of IDS - VDS . We have also explored the device scaling effect on the amplifier gain and found that ~ 2.23× gain can be increased with smaller channel length and higher device width.
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Till date, the existing understanding of negative differential resistance (NDR) is obtained from metal-ferro-metal-insulator-semiconductor (MFMIS) FET, and it has been utilized for both MFMIS and metal-ferro-insulator-semiconductor (MFIS) based NCFETs. However, in MFIS architecture, the ferroelectric capacitance (CFE) is not a lumped capacitance. Therefore, for MFIS negative capacitance (NC) devices, the physical explanation which governs the NDR mechanism needs to be addressed. In this work, for the first time, we present the first principle explanation of the NDR effect in MFIS NC FDSOI. We found that the output current variation with the drain to source voltage (VDS), (i.e.gds) primarily depends upon two parameters: (a)VDSdependent inversion charge gradient (∂n/∂VDS); (b)VDSsensitive electron velocity (∂v/∂VDS), and the combined effect of these two dependencies results in NDR. Further, to mitigate the NDR effect, we proposed the BOX engineered NC FDSOI FET, in which the buried oxide (BOX) layer is subdivided into the ferroelectric (FE) layer and the SiO2layer. In doing so, the inversion charge in the channel is enhanced by the BOX engineered FE layer, which in turn mitigates the NDR and a nearly zerogdswith a minimal positive slope has been obtained. Through well-calibrated TCAD simulations, by utilizing the obtained positivegds, we also designed aVDSindependent constant current mirror which is an essential part of analog circuits. Furthermore, we discussed the impact of the FE parameter (remanent polarization and coercive field) variation on the device performances. We have also compared the acquired results with existing literature on NC-based devices, which justifies that our proposed structure exhibits complete diminution of NDR, thus enabling its use in analog circuit design.
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Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein aquaporin-2. Vasopressin signaling is initiated by binding to a G-protein-coupled receptor called V2R, which signals through heterotrimeric G-protein subunit Gs α, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteome-wide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2R-selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad-spectrum kinase inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide), and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic data sets have been made available online for modeling vasopressin signaling and signaling downstream from other G-protein-coupled receptors. SIGNIFICANCE STATEMENT: New developments in protein mass spectrometry are facilitating progress in identification of signaling networks. Using mass spectrometry, it is now possible to identify and quantify thousands of phosphorylation sites in a given cell type (phosphoproteomics). The authors describe the use of phosphoproteomics technology to identify signaling mechanisms downstream from a G-protein-coupled receptor, the vasopressin V2 subtype receptor, and its role of the regulation and dysregulation of water excretion in the kidney. Data from multiple phosphoproteomic data sets are provided as web-based resources.
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Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Rim/metabolismo , Fosforilação/fisiologia , Proteoma/metabolismo , Transdução de Sinais/fisiologia , Vasopressinas/metabolismo , Animais , Humanos , Proteômica/métodosRESUMO
BACKGROUND AND PURPOSE: The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a PKA-dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. EXPERIMENTAL APPROACH: We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method). KEY RESULTS: Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk). CONCLUSION AND IMPLICATIONS: In V2 receptor-mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web-based data resource for future studies of GPCRs.
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Túbulos Renais Coletores , Proteínas Quinases , Vasopressinas , Animais , Aquaporina 2/metabolismo , Túbulos Renais Coletores/metabolismo , Camundongos , Fosforilação , Proteínas Quinases/metabolismo , Proteoma , Vasopressinas/metabolismoRESUMO
Cells assemble microns-long filamentous structures from protein monomers that are nanometers in size. These structures are often highly dynamic, yet in order for them to function properly, cells maintain them at a precise length. Here we investigate length-dependent depolymerization as a mechanism of length control. This mechanism has been recently proposed for flagellar length control in the single cell organisms Chlamydomonas and Giardia. Length dependent depolymerization can arise from a concentration gradient of a depolymerizing protein, such as kinesin-13 in Giardia, along the length of the flagellum. Two possible scenarios are considered: a linear and an exponential gradient of depolymerizing proteins. We compute analytically the probability distributions of filament lengths for both scenarios and show how these distributions are controlled by key biochemical parameters through a dimensionless number that we identify. In Chlamydomonas cells, the assembly dynamics of its two flagella are coupled via a shared pool of molecular components that are in limited supply, and so we investigate the effect of a limiting monomer pool on the length distributions. Finally, we compare our calculations to experiments. While the computed mean lengths are consistent with observations, the noise is two orders of magnitude smaller than the observed length fluctuations.
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Flagelos/metabolismo , Polimerização , Transporte Biológico , Chlamydomonas/metabolismo , Giardia/metabolismo , Cinesinas/metabolismoRESUMO
Vasopressin regulates renal water excretion by binding to a Gα s-coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin-2 (AQP2) water channel. This action is widely accepted to be associated with cAMP-mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR-Cas9) to identify PKA-independent responses to vasopressin. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Upregulated sites in PKA-null cells include Ser256 of AQP2, which is critical to regulation of AQP2 trafficking. In addition, phosphorylation changes in the protein kinases Stk39 (SPAK) and Prkci (an atypical PKC) are consistent with PKA-independent regulation of these protein kinases. Target motif analysis of the phosphopeptides increased in PKA-null cells indicates that vasopressin activates one or more members of the AMPK/SNF1-subfamily of basophilic protein kinases. In vitro phosphorylation assays using recombinant, purified SNF1-subfamily kinases confirmed postulated target specificities. Of interest, measured IBMX-dependent cAMP levels were an order of magnitude higher in PKA-null than in PKA-intact cells, indicative of a PKA-dependent feedback mechanism. Overall, the findings support the conclusion that V2-receptor mediated signaling in collecting duct cells is in part PKA-independent.
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Aquaporina 2/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Túbulos Renais Coletores/metabolismo , Fosfoproteínas/metabolismo , Proteoma/análise , Receptores de Vasopressinas/metabolismo , Animais , Túbulos Renais Coletores/citologia , Camundongos , FosforilaçãoRESUMO
Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.
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Aquaporina 2/metabolismo , Túbulos Renais Coletores/metabolismo , Fosfoproteínas/metabolismo , Receptores de Vasopressinas/metabolismo , Aminoácidos/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Medula Renal/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Fármacos Renais/farmacologia , Transdução de Sinais , Vasopressinas/farmacologiaRESUMO
Lewy body dementias are the second most common cause of neurodegenerative dementia in the elderly after Alzheimer's disease (AD). The two clinical subgroups of Lewy body dementias, namely, dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), are differentiated by the chronology of cognitive symptoms relative to parkinsonism. At present, there remains a debate on whether DLB and PDD are separate disease entities, or fall within the same spectrum of Lewy body dementias. In this study, we compared the detergent-soluble proteome via an 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) analysis of pooled lysates from the prefrontal cortex (BA9) of DLB (n = 19) and PDD (n = 21) patients matched a priori for amyloid (total Aß42) burden, semi-quantitative scores for Lewy bodies and neurofibrillary tangles together with age-matched control (n = 21) subjects. A total of 1914 proteins were confidently identified by iTRAQ (false discovery rate = 0%). None of the proteins showed a significant yet opposite regulation in between DLB and PDD when compared to aged controls in the proteomic data set as well as following immunoblot analysis of the pooled and individual lysates involving all 61 subjects. The postsynaptic protein, synaptopodin (SYNPO) was significantly down-regulated in both DLB and PDD subgroups, suggesting a defective synaptic transmission in the demented patients. In conclusion, the largely similar proteome of DLB and PDD matched for amyloid burden suggests that variations in concomitant AD-related pathology, abnormal post-translational modifications or protein-protein interactions, defective intracellular trafficking or misfolding of proteins could play a part in driving the clinically observed differences between these two subgroups of Lewy body dementias. This further indicates that amyloid-targeting therapeutic strategies may show different efficacies in DLB versus PDD.
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Marcação por Isótopo , Doença por Corpos de Lewy/metabolismo , Neocórtex/metabolismo , Proteômica/métodos , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Demografia , Feminino , Humanos , Immunoblotting , Doença por Corpos de Lewy/patologia , Masculino , Proteínas dos Microfilamentos , Neocórtex/patologia , Controle de QualidadeRESUMO
Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. One probable explanation lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicate that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses reveal that SF3B1 specifically binds nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affects splicing of these exons similarly to SF3B1 knockdown. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice-site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure.
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Nucleossomos/metabolismo , Fosfoproteínas/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Sequência de Bases , Éxons , Sequência Rica em GC , Células HeLa , Humanos , Dados de Sequência Molecular , Nucleossomos/genética , Fosfoproteínas/genética , Ligação Proteica , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/genéticaRESUMO
Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.