RESUMEN
There is heritability to interindividual variation in platelet count, and better understanding of the regulating genetic factors may provide insights for thrombopoiesis. MicroRNAs (miRs) regulate gene expression in health and disease, and megakaryocytes (MKs) deficient in miRs have lower platelet counts, but information about the role of miRs in normal human MK and platelet production is limited. Using genome-wide miR profiling, we observed strong correlations among human bone marrow MKs, platelets, and differentiating cord blood-derived MK cultures, and identified MK miR-125a-5p as associated with human platelet number but not leukocyte or hemoglobin levels. Overexpression and knockdown studies showed that miR-125a-5p positively regulated human MK proplatelet (PP) formation in vitro. Inhibition of miR-125a-5p in vivo lowered murine platelet counts. Analyses of MK and platelet transcriptomes identified LCP1 as a miR-125a-5p target. LCP1 encodes the actin-bundling protein, L-plastin, not previously studied in MKs. We show that miR-125a-5p directly targets and reduces expression of MK L-plastin. Overexpression and knockdown studies show that L-plastin promotes MK progenitor migration, but negatively correlates with human platelet count and inhibits MK PP formation (PPF). This work provides the first evidence for the actin-bundling protein, L-plastin, as a regulator of human MK PPF via inhibition of the late-stage MK invagination system, podosome and PPF, and PP branching. We also provide resources of primary and differentiating MK transcriptomes and miRs associated with platelet counts. miR-125a-5p and L-plastin may be relevant targets for increasing in vitro platelet manufacturing and for managing quantitative platelet disorders.
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Plaquetas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Megacariocitos/citología , Megacariocitos/metabolismo , Glicoproteínas de Membrana/genética , MicroARNs/genética , Proteínas de Microfilamentos/genética , Trombopoyesis/genética , Actinas/metabolismo , Biomarcadores , Técnicas de Silenciamiento del Gen , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Interferencia de ARNRESUMEN
Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2 BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbß3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.
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Proteínas Reguladoras de la Apoptosis/biosíntesis , Sangre Fetal , Megacariocitos , Antígenos de Diferenciación/biosíntesis , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Megacariocitos/citología , Megacariocitos/metabolismoRESUMEN
Comprehending the decomposition process is crucial for our understanding of the mechanisms of carbon (C) sequestration in soils. The decomposition of plant biomass has been extensively studied. It revealed that extrinsic biomass properties that restrict its access to decomposers influence decomposition more than intrinsic ones that are only related to its chemical structure. Fungal biomass has been much less investigated, even though it contributes to a large extent to soil organic matter, and is characterized by specific biochemical properties. In this study, we investigated the extent to which decomposition of heathland fungal biomass was affected by its hydrophobicity (extrinsic property) and melanin content (intrinsic property). We hypothesized that, as for plant biomass, hydrophobicity would have a greater impact on decomposition than melanin content. Mineralization was determined as the mineralization of soil organic carbon (SOC) into CO2 by headspace GC/MS after inoculation by a heathland soil microbial community. Results show that decomposition was not affected by hydrophobicity, but was negatively correlated with melanin content. We argue that it may indicate that either melanin content is both an intrinsic and extrinsic property, or that some soil decomposers evolved the ability to use surfactants to access to hydrophobic biomass. In the latter case, biomass hydrophobicity should not be considered as a crucial extrinsic factor. We also explored the ecology of decomposition, melanin content, and hydrophobicity, among heathland soil fungal guilds. Ascomycete black yeasts had the highest melanin content, and hyaline Basidiomycete yeasts the lowest. Hydrophobicity was an all-or-nothing trait, with most isolates being hydrophobic.
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Hongos/crecimiento & desarrollo , Melaninas/análisis , Microbiología del Suelo , Suelo/química , Biomasa , Ecosistema , Hongos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Plantas/química , Plantas/metabolismoRESUMEN
The last decade has experienced the emergence of microRNAs as a key molecular tool for the diagnosis and prognosis of human diseases. Although the focus has mostly been on cancer, neurodegenerative diseases present an exciting, yet less explored, platform for microRNA research. Several studies have highlighted the significance of microRNAs in neurogenesis and neurodegeneration, and pre-clinical studies have shown the potential of microRNAs as biomarkers. Despite this, no bona fide microRNAs have been identified as true diagnostic or prognostic biomarkers for neurodegenerative disease. This is mainly due to the lack of precisely defined patient cohorts and the variability within and between individual cohorts. However, the discovery that microRNAs exist as stable molecules at detectable levels in body fluids has opened up new avenues for microRNAs as potential biomarker candidates. Furthermore, technological developments in microRNA biology have contributed to the possible design of microRNA-mediated disease intervention strategies. The combination of these advancements, with the availability of well-defined longitudinal patient cohort, promises to not only assist in developing invaluable diagnostic tools for clinicians, but also to increase our overall understanding of the underlying heterogeneity of neurodegenerative diseases. In this review, we present a comprehensive overview of the existing knowledge of microRNAs in neurodegeneration and provide a perspective of the applicability of microRNAs as a basis for future therapeutic intervention strategies.
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MicroARNs/genética , MicroARNs/uso terapéutico , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/terapia , Animales , Autofagia , Encéfalo/metabolismo , Encéfalo/patología , Descubrimiento de Drogas , Regulación de la Expresión Génica , Marcadores Genéticos , Terapia Genética , Humanos , MicroARNs/análisis , MicroARNs/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Transcripción GenéticaRESUMEN
Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that â¼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.
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Anomalías Múltiples/genética , Anomalías Múltiples/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Enfermedades Neurodegenerativas/genética , Neuronas/patología , Proteínas de Pez Cebra/genética , Sinucleína beta/genética , Secuencia de Aminoácidos , Animales , Química Encefálica/genética , Neuronas Dopaminérgicas , Técnicas de Silenciamiento del Gen , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/biosíntesis , Locomoción , Mutación/genética , Enfermedades Neurodegenerativas/patología , Enfermedad de Parkinson/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Pez Cebra , Proteínas de Pez Cebra/biosíntesisRESUMEN
Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration.
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Adaptación Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Calor , Serina Proteasas/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Western Blotting , Clonación Molecular , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Proteínas Oncogénicas/química , Proteínas Oncogénicas/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Plantas Modificadas Genéticamente , Proteína Desglicasa DJ-1 , Desplegamiento Proteico , Proteolisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Serina Proteasas/metabolismo , Especificidad por Sustrato , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive and irreversible neurodegenerative disorder coupled to selective degeneration of dopamine-producing neurons in the substantia nigra. The majority of PD incidents are sporadic, but monogenic cases account for 5-10% of cases. Mutations in PINK1 cause autosomal recessive forms of early-onset PD, and PINK1 stimulates Omi/HtrA2/PARK13 protease activity when both proteins act as neuroprotective components in the same stress pathway. Studies on PINK1 and PARK13 have concentrated on phosphorylation-dependent PINK1-mediated activation of PARK13 and mitochondrial functions, because both proteins are classically viewed as mitochondrial. Although PARK13-mediated protective mechanisms are at least in part regulated by PINK1, little is known concerning how these two proteins are regulated in different subcellular compartments or, indeed, the influence of PARK13 on PINK1 characteristics. We show that PARK13 localizes to a variety of subcellular locations in neuronal cells and that PINK1, although more restrictive, also localizes to locations other than those previously reported. We demonstrate that PARK13 accumulation leads to a concomitant accumulation of PINK1 and that the increase in PINK1 levels is compartmental specific, indicating a correlative relationship between the two proteins. Moreover, we show that PARK13 and PINK1 protein levels accumulate in response to H2 O2 and L-DOPA treatments in a subcellular fashion and that both proteins show relocation to the cytoskeleton in response to H2 O2 . This H2 O2 -mediated relocation is abolished by PARK13 overexpression. This study shows that PARK13 and PINK1 are subcellular-specific, but dynamic, proteins with a reciprocal molecular relationship providing new insight into the complexity of PD.
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Neuronas/citología , Estrés Oxidativo/fisiología , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/metabolismo , Fracciones Subcelulares/metabolismo , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Dopaminérgicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Peróxido de Hidrógeno/farmacología , Levodopa/farmacología , Mutación/genética , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/genética , Proteínas Quinasas/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.
Asunto(s)
Diferenciación Celular , Neuronas Dopaminérgicas , Células Madre Pluripotentes Inducidas , Factores de Transcripción , Humanos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Factores de Transcripción/metabolismo , Lentivirus/genética , Lentivirus/metabolismoRESUMEN
Plastids are complex organelles that are integrated into the plant host cell where they differentiate and divide in tune with plant differentiation and development. In line with their prokaryotic origin, plastid division involves both evolutionary conserved proteins and proteins of eukaryotic origin where the host has acquired control over the process. The plastid division apparatus is spatially separated between the stromal and the cytosolic space but where clear coordination mechanisms exist between the two machineries. Our knowledge of the plastid division process has increased dramatically during the past decade and recent findings have not only shed light on plastid division enzymology and the formation of plastid division complexes but also on the integration of the division process into a multicellular context. This review summarises our current knowledge of plastid division with an emphasis on biochemical features, the functional assembly of protein complexes and regulatory features of the overall process.
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Proteínas de Arabidopsis/química , División Celular , Plastidios/química , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diferenciación Celular , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Citosol/química , Evolución Molecular , Membranas Intracelulares/química , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Fosforilación , Células Vegetales/química , Plastidios/genéticaRESUMEN
Besides respiratory illness, SARS-CoV-2, the causative agent of COVID-19, leads to neurological symptoms. The molecular mechanisms leading to neuropathology after SARS-CoV-2 infection are sparsely explored. SARS-CoV-2 enters human cells via different receptors, including ACE-2, TMPRSS2, and TMEM106B. In this study, we used a human-induced pluripotent stem cell-derived neuronal model, which expresses ACE-2, TMPRSS2, TMEM106B, and other possible SARS-CoV-2 receptors, to evaluate its susceptibility to SARS-CoV-2 infection. The neurons were exposed to SARS-CoV-2, followed by RT-qPCR, immunocytochemistry, and proteomic analyses of the infected neurons. Our findings showed that SARS-CoV-2 infects neurons at a lower rate than other human cells; however, the virus could not replicate or produce infectious virions in this neuronal model. Despite the aborted SARS-CoV-2 replication, the infected neuronal nuclei showed irregular morphology compared to other human cells. Since cytokine storm is a significant effect of SARS-CoV-2 infection in COVID-19 patients, in addition to the direct neuronal infection, the neurons were treated with pre-conditioned media from SARS-CoV-2-infected lung cells, and the neuroproteomic changes were investigated. The limited SARS-CoV-2 infection in the neurons and the neurons treated with the pre-conditioned media showed changes in the neuroproteomic profile, particularly affecting mitochondrial proteins and apoptotic and metabolic pathways, which may lead to the development of neurological complications. The findings from our study uncover a possible mechanism behind SARS-CoV-2-mediated neuropathology that might contribute to the lingering effects of the virus on the human brain.
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COVID-19 , Humanos , SARS-CoV-2 , Medios de Cultivo Condicionados , Proteómica , Redes y Vías Metabólicas , Proteínas de la Membrana , Proteínas del Tejido NerviosoRESUMEN
Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.
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Neutrófilos , Receptores de Trombina , Animales , Catepsina G , Perros , Ligandos , Ratones , Neutrófilos/metabolismo , Proteolisis , Ratas , Receptores de Trombina/metabolismoRESUMEN
Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement-a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.
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Proteínas de Membrana de los Lisosomas/genética , Enfermedades Neurodegenerativas/genética , Lipofuscinosis Ceroideas Neuronales/genética , Neuronas/metabolismo , Adolescente , Adulto , Sistemas CRISPR-Cas/genética , Catepsina B/farmacología , Línea Celular , Corteza Cerebelosa/crecimiento & desarrollo , Corteza Cerebelosa/metabolismo , Niño , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de Membrana de los Lisosomas/antagonistas & inhibidores , Lisosomas/genética , Mutación/genética , Enfermedades Neurodegenerativas/complicaciones , Enfermedades Neurodegenerativas/fisiopatología , Lipofuscinosis Ceroideas Neuronales/complicaciones , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/patología , Fenotipo , Adulto JovenRESUMEN
This paper presents the formulation, inkjet printing, and vacuum forming of a conductive and stretchable polymer, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), ink on a stretchable and transparent thermoplastic polyurethane (TPU) substrate. The formulation of the conductive and stretchable ink is achieved by combining PEDOT:PSS with additional solvents, to achieve the right inkjet properties for drop-on-demand (DoD) inkjet printing. A conductive pattern can be printed from the 21 µm orifice on a flexible and stretchable TPU substrate, with a linewidth down to 44 µm. The properties of the printed pattern, in terms of sheet resistance, morphology, transparency, impact of weather conditions, and stretching are investigated and show sheet resistances up to 45 Ohm/sq and transparencies as high as 95%, which is comparable to indium tin oxide (ITO). Moreover, in contrast to ITO, one-time stretching up to 40% can be achieved, increasing the sheet resistance up to 214 Ohm/sq only, showing the great potential of this ink for one-time stretching. Finally, as a proof of this one-time stretching, the printed samples are vacuum formed around a 3D object, still showing sufficient conductivity to be applied as a capacitive touch sensor.
RESUMEN
Essentials The action of microRNAs (miRs) in human megakaryocyte signaling is largely unknown. Cord blood-derived human megakaryocytes (MKs) were used to test the function of candidate miRs. miR-15a-5p negatively regulated MK GPVI-mediated αIIbß3 activation and α-granule release. miR-15a-5p acts as a potential "master-miR" regulating genes in the MK GPVI signaling pathway. SUMMARY: Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhibit mRNA translation into protein, are abundantly expressed in MKs and platelets. Although platelet miRs have been associated with platelet reactivity and disease, there is a paucity of information on the function of miRs in human MKs. Objective To identify MK miRs that regulate the GPVI signaling pathway in the MK-platelet lineage. Methods Candidate miRs associated with GPVI-mediated platelet aggregation were tested for functionality in cultured MKs derived from cord blood. Results An unbiased, transcriptome-wide screen in 154 healthy donors identified platelet miR-15a-5p as significantly negatively associated with CRP-induced platelet aggregation. Platelet agonist dose-response curves demonstrated activation of αIIbß3 in suspensions of cord blood-derived cultured MKs. Overexpression and knockdown of miR-15a-5p in these MKs reduced and enhanced, respectively, CRP-induced αIIbß3 activation but did not alter thrombin or ADP stimulation. FYN, SRGN, FCER1G, MYLK. and PRKCQ, genes involved in GPVI signaling, were identified as miR-15a-5p targets and were inhibited or de-repressed in MKs with miR-15a-5p overexpression or inhibition, respectively. Lentiviral overexpression of miR-15a-5p also inhibited GPVI-FcRγ-mediated phosphorylation of Syk and PLCγ2, GPVI downstream signaling molecules, but effects of miR-15a-5p on αIIbß3 activation did not extend to other ITAM-signaling receptors (FcγRIIa and CLEC-2). Conclusion Cord blood-derived MKs are a useful human system for studying the functional effects of candidate platelet genes. miR-15a-5p is a potential "master-miR" for specifically regulating GPVI-mediated MK-platelet signaling. Targeting miR-15a-5p may have therapeutic potential in hemostasis and thrombosis.
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Plaquetas/metabolismo , Megacariocitos/metabolismo , MicroARNs/metabolismo , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Transducción de Señal , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Sangre Fetal/citología , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , MicroARNs/genética , Activación Plaquetaria/genética , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Transducción de Señal/genéticaRESUMEN
INTRODUCTION: As current clinical diagnostic protocols for Parkinson's disease (PD) may be prone to inaccuracies there is a need to identify and validate molecular biomarkers, such as circulating microRNAs, which will complement current practices and increase diagnostic accuracy. This study identifies, verifies and validates combinatory serum microRNA signatures as diagnostic classifiers of PD across different patient cohorts. METHODS: 370 PD (drug naïve) and control serum samples from the Norwegian ParkWest study were used for identification and verification of differential microRNA levels in PD which were validated in a blind study using 64 NY Parkinsonism in UMeå (NYPUM) study serum samples and tested for specificity in 48 Dementia Study of Western Norway (DemWest) study Alzheimer's disease (AD) serum samples using miRNA-microarrays, and quantitative (q) RT-PCR. Proteomic approaches identified potential molecular targets for these microRNAs. RESULTS: Using Affymetrix GeneChip® miRNA 4.0 arrays and qRT-PCR we comprehensively analyzed serum microRNA levels and found that the microRNA (PARKmiR)-combinations, hsa-miR-335-5p/hsa-miR-3613-3p (95% CI, 0.87-0.94), hsa-miR-335-5p/hsa-miR-6865-3p (95% CI, 0.87-0.93), and miR-335-5p/miR-3613-3p/miR-6865-3p (95% CI, 0.87-0.94) show a high degree of discriminatory accuracy (AUC 0.9-1.0). The PARKmiR signatures were validated in an independent PD cohort (AUCâ¯≤â¯0.71) and analysis in AD serum samples showed PARKmiR signature specificity to PD. Proteomic analyses showed that the PARKmiRs regulate key PD-associated proteins, including alpha-synuclein and Leucine Rich Repeat Kinase 2. CONCLUSIONS: Our study has identified and validated unique miRNA serum signatures that represent PD classifiers, which may complement and increase the accuracy of current diagnostic protocols.
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MicroARNs/sangre , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/diagnóstico , Anciano , Enfermedad de Alzheimer/sangre , Biomarcadores/sangre , Estudios de Cohortes , Femenino , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Noruega , Análisis de Secuencia de ARN , SueciaRESUMEN
Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein α-synuclein. α-synuclein is considered an intrinsically disordered 14 kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that α-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different α-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa α-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific α-synuclein features in neurodegeneration will allow for the identification of neurotoxic α-synuclein species, which represent prime targets to reduce α-synuclein pathogenicity.
Asunto(s)
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Degeneración Nerviosa/metabolismo , Enfermedad de Parkinson/metabolismo , Fracciones Subcelulares/metabolismo , Encéfalo/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Núcleo Celular/química , Cromatografía Líquida de Alta Presión/métodos , Citosol/química , Humanos , Fracciones Subcelulares/químicaRESUMEN
Parkinson's disease is a chronic, progressive neurodegenerative disorder with increased prevalence in the aging population. It is estimated that approximately 1.5 million individuals in the US alone suffer from Parkinson's disease and with the extension of life expectancy this number is expected to rise dramatically within the next twenty-five years. The majority of Parkinson's disease cases are sporadic. But mutations in genes such as α-synuclein, Parkin, PINK1, DJ-1 and LRRK2, have been conclusively associated with both early- and late-onset of the disease. Although the genetics of Parkinson's disease is starting to become unraveled, the interplay between genetic and environmental factors is largely unknown as are the underlying mechanisms that trigger the disease as the brain ages. The risk of Parkinson's disease increases dramatically in individuals over the age of 60 and it is estimated that more than 1% of all seniors have some form of the condition. In this review, we will highlight some of the central proteins associated with Parkinson's disease and how they may be linked to processes and factors associated with age.
Asunto(s)
Envejecimiento/fisiología , Enfermedad de Parkinson/etiología , Autofagia/fisiología , Muerte Celular/fisiología , Ambiente , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Enfermedades de Inicio Tardío/etiología , Enfermedades de Inicio Tardío/fisiopatología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mitofagia/fisiología , Neuronas/fisiología , Proteínas Oncogénicas/fisiología , Estrés Oxidativo/fisiología , Enfermedad de Parkinson/fisiopatología , Proteína Desglicasa DJ-1 , Proteínas Serina-Treonina Quinasas/fisiología , alfa-Sinucleína/fisiologíaRESUMEN
MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson's disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant (p-value < 0.05) changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells.