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Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.
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Anticuerpos Monoclonales , Histidina/metabolismo , Animales , Centrosoma , Cromatografía Liquida , Células HeLa , Humanos , Modelos Químicos , Péptidos/análisis , Fosforilación , Polos del Huso , Espectrometría de Masas en TándemRESUMEN
The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.
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Células Dendríticas/inmunología , Macrófagos/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Animales , Apoptosis/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Immunoblotting , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Rastreo , Fagocitosis/inmunología , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson's disease. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.
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Encéfalo/metabolismo , Microglía/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis , Encéfalo/irrigación sanguínea , Encéfalo/citología , Encéfalo/patología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Modelos Animales de Enfermedad , Femenino , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Masculino , Ratones , Neurogénesis , Enfermedad de Parkinson/metabolismo , Fagocitosis , Proteína S/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Tirosina Quinasas Receptoras/deficiencia , Transducción de Señal , Nicho de Células Madre , Regulación hacia Arriba , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty acid ß-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in patients with NASH; however, the effect of ACC inhibition on liver fibrosis has not been reported. METHODS: A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, diethylnitrosamine. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. RESULTS: Inhibition of ACC reduced the activation of TGF-ß-stimulated HSCs, as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which inhibition of de novo lipogenesis blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require de novo lipogenesis for activation. Consistent with this direct antifibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat choline-deficient, high-fat diet model and in response to chronic diethylnitrosamine-induced liver injury (in the absence of hepatic lipid accumulation). CONCLUSIONS: In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the profibrogenic activity of HSCs. Thus, small molecule inhibitors of ACC may lessen fibrosis by reducing lipotoxicity in hepatocytes and by preventing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH. LAY SUMMARY: Hepatic fibrosis is the most important predictor of liver-related outcomes in patients with non-alcoholic steatohepatitis (NASH). Small molecule inhibitors of acetyl-CoA carboxylase (ACC) reduce hepatic fat content and markers of liver injury in patients with NASH. Herein, we report that inhibition of ACC and de novo lipogenesis also directly suppress the activation of hepatic stellate cells - the primary cell responsible for generating fibrotic scar in the liver - and thus fibrosis. These data provide further evidence for the use of ACC inhibitors to treat patients with NASH and advanced fibrosis.
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Acetil-CoA Carboxilasa/antagonistas & inhibidores , Células Estrelladas Hepáticas/metabolismo , Lipogénesis/efectos de los fármacos , Cirrosis Hepática/metabolismo , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Humanos , Hígado/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Ratas , Ratas WistarRESUMEN
Fluoroscopy is increasingly used to guide minimally invasive endourological procedures and optimised protocols are needed to minimise radiation exposure while achieving best treatment results. This multi-center study of radiation exposure of patients was conducted by the South-Eastern European Group for Urolithiasis Research (SEGUR), in cooperation with the International Atomic Energy Agency. Seven clinical centers from the SEGUR group collected data for 325 procedures performed within a three-months period, including standard percutaneous nephrolithotomy (PCNL), mini PCNL, retrograde intrarenal surgery (RIRS), semirigid ureterorenoscopy (URS) and flexible URS. Data included: air kerma area product (PKA), air kerma at the patient entrance reference point (Ka,r), fluoroscopy time (FT), number of radiographic images (N) and fluoroscopy pulse rate, as well as total procedure duration, size and location of stones. Data were centrally analysed and statistically compared. MedianPKAvalues per center varied 2-fold for RIRS (0.80-1.79 Gy cm2), 7.1 fold for mini-PCNL (1.39-9.90 Gy cm2), 7.3 fold for PCNL (2.40-17.50 Gy cm2), 19 fold (0.13-2.51 Gy cm2) for semi-rigid URS and 29-fold for flexible URS (0.10-2.90 Gy cm2). LowerPKAandKa,rwere associated with use of lower FT,Nand lower fluoroscopy pulse rate. FT varied from 0.1 to 14 min, a small fraction of the total procedure time, ranging from 10 to 225 min. HigherNwas associated with higherPKAandKa,r. Higher medianPKAin PCNL was associated with the use of supine compared to prone position. No correlation was found between the concrement size and procedure duration, FT,PKAorKa,r. Dose values for RIRS were significantly lower compared to PCNL. The maximumKa,rvalue of 377 mGy was under the threshold for radiation induced skin erythema. The study demonstrated a potential for patient dose reduction by lowering FT andN, using pulsed fluoroscopy and beam collimation.
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Exposición a la Radiación , Urolitiasis , Fluoroscopía/efectos adversos , Humanos , Riñón , Resultado del TratamientoRESUMEN
NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCFßTrCP (Skp, Cullin and F-boxßTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser476 and Ser480). This induces binding of NUAK1 to ßTrCP, the substrate-recognition subunit of the SCFßTrCP E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G2-M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1ßMYPT1 (where PP1 is protein phosphatase 1) and that a major role for the PP1ßMYPT1 complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr210). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFßTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1ßMYPT1 phosphatase.
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Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Fosfatasa de Miosina de Cadena Ligera/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina-Proteína Ligasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Alineación de Secuencia , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Quinasa Tipo Polo 1RESUMEN
Due to the intricate and volatile nature of the service environment surrounding prestressing anchoring materials, stress corrosion poses a significant challenge to the sustained stability of underground reinforcement systems. Consequently, it is imperative to identify effective countermeasures against stress corrosion failure in cable bolts within deep underground environments, thereby ensuring the safety of deep resource extraction processes. In this study, the influence of various coatings on the stress corrosion resistance of cable bolts was meticulously examined and evaluated using specifically designed stress-corrosion-testing systems. The specimens were subjected to loading using four-point bending frames and exposed to simulated underground corrosive environments. A detailed analysis and comparison of the failure patterns and mechanisms of specimens coated with different materials were conducted through the meticulous observation of fractographic features. The results revealed stark differences in the stress corrosion behavior of coated and uncoated bolts. Notably, epoxy coatings and chlorinated rubber coatings exhibited superior anti-corrosion capabilities. Conversely, galvanized layers demonstrated the weakest effect due to their sacrificial anti-corrosion mechanism. Furthermore, the effectiveness of the coatings was found to be closely linked to the curing agent and additives used. The findings provide valuable insights for the design and selection of coatings that can enhance the durability and reliability of cable bolts in deep underground environments.
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When modelling the anchorage performance of rebars with the tri-linear law, the calculation process of the load-deformation relation is complicated. The reason is that when the rebar-grout interface entered the elastic-softening-debonding stage, the softening section length and debonding section length vary simultaneously. To solve this issue, this paper proposes an enhanced numerical calculation method. When the rebar-grout interface entered the elastic-softening-debonding stage, the softening section length was fixed to a specific value. One loop function was created to calculate the debonding section length. With this method, the number of iteration calculations significantly decreased. The credibility of this calculation method was confirmed with experimental results. Two case studies were conducted to compare the load-deformation relation obtained with the original calculation method and enhanced calculation method. The results showed that good consistency existed between the results obtained by those two methods. This finding can significantly improve the calculation efficiency when studying the anchorage performance of rebars. Moreover, this paper provides new insight for users to optimise the modelling process of rebars.
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The grout annulus (GA) has a significant effect on the tensile performance of rockbolts in mining engineering. However, little research has been conducted to use modified cable elements to study this effect quantitatively. This paper used the modified cable elements in FLAC3D to study the effect of the GA on the tensile performance of rockbolts. The two-stage coupling law was used to simulate the behaviour of the GA. The stress had a linear relation with the slippage before the shear strength (SS). After the SS, the stress decreased exponentially. Numerical in situ roadway reinforcement cases were used to study the influence of the grout annulus on the tensile performance of rockbolts. The results showed that, when the SS of the GA increased from 3.2 MPa to 6.4 Mpa, the peak force of rockbolts increased from 247 kN to 425 kN. Moreover, when the SS of the GA increased from 3.2 Mpa to 6.4 Mpa, the distance between the position of the maximum tensile capacity and the external end decreased from 1.17 m to 0.81 m. Last, for the circular roadway, the peak force in rockbolts installed in the lateral side was 171.7 kN, which was significantly larger than the top side of 72.3 kN.
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This study proposes utilising the solution blow-spinning process (SBS) for manufacturing a biodegradable filtration structure that ensures high efficiency of particle filtration with an acceptable pressure drop. The concept of multi-layer filters was applied during the design of filters. Polylactic acid (PLA) was used to produce various layers, which may be mixed in different sequences, building structures with varying filtration properties. Changing the process parameters, one can create layers with diverse average fibre diameters and thicknesses. It enables the design and creation of optimal filtration materials prepared for aerosol particle filtration. The structures were numerically modelled using the lattice Boltzmann approach to obtain detailed production guidelines using the blow-spinning technique. The advantage of this method is the ability to blow fibres with diameters in the nanoscale, applying relatively simple and cost-effective equipment. For tested PLA solutions, i.e., 6% and 10%, the mean fibre diameter decreases as the concentration decreases. Therefore, the overall filtering efficiency decreases as the concentration of the used solution increases. The produced multi-layer filters have 96% overall filtration efficiency for particles ranging from 0.26 to 16.60 micrometres with a pressure drop of less than 160 Pa. Obtained results are auspicious and are a step in producing efficient, biodegradable air filters.
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Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.
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Proteínas Serina-Treonina Quinasas/metabolismo , Sorbitol/farmacología , Secuencia de Aminoácidos , Animales , Dominio Catalítico/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Clatrina/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/enzimología , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Presión Osmótica , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Transporte de Proteínas/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
BACKGROUND AND PURPOSE: Cerebral artery fenestrations (CAF) are rare congenital variations usually diagnosed by digital subtraction angiography (DSA). The aim of this study was to examine the frequency of occurrence of fenestrations in cerebral arteries and their coexistence with cerebral aneurysms in computed tomography angiography (CTA). MATERIAL AND METHODS: All reports of cerebral CTA (1140) performed in one institution from March 2005 to December 2007 were analysed. We found 40 patients with single fenestrations of the intracranial arteries. All 40 examinations were retrospectively reviewed for location of vascular malformations and presence of aneurysms or subarachnoid haemorrhage (SAH). Medical histories of those patients were then analysed for evidence of SAH and referral reasons for CTA. RESULTS: Forty fenestrated arteries were found in CTA: 18 basilar arteries (45%), 16 anterior cerebral arteries (40%), 4 anterior communicating arteries (10%) and one middle cerebral artery (2.5%). Only one vertebral artery fenestration was found due to the technique of the examination. Six patients (15%) with fenestrated arteries had a total of 8 aneurysms, although only one aneurysm was ipsilateral to the fenestration. In 8 cases of SAH, two were with no evidence of vascular malformation. The coexistence of CAF and aneurysms in CTA amounted to 15% (6/40), but the incidence of ipsilateral aneurysm was only 2.5% (1/40) and it affected the anterior cerebral artery. CONCLUSIONS: Basilar artery fenestration is the most frequent observed fenestration in CTA, followed by anterior cerebral artery and anterior communicating artery fenestrations. Coexistence of fenestration and aneurysm is uncommon in CTA examination.
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Arteria Cerebral Anterior/anomalías , Arteria Cerebral Anterior/diagnóstico por imagen , Arteria Carótida Interna/anomalías , Arteria Carótida Interna/cirugía , Malformaciones Vasculares del Sistema Nervioso Central/diagnóstico por imagen , Aneurisma Intracraneal/diagnóstico por imagen , Adolescente , Adulto , Anciano , Arteria Cerebral Anterior/patología , Arteria Carótida Interna/patología , Malformaciones Vasculares del Sistema Nervioso Central/patología , Angiografía Cerebral , Femenino , Humanos , Aneurisma Intracraneal/patología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tomografía Computarizada por Rayos XRESUMEN
Small ubiquitin-like modifier (SUMO)-specific protease SENP1 processes SUMO-1, SUMO-2 and SUMO-3 to mature forms and deconjugates them from modified proteins. To establish the proteolytic mechanism, we determined structures of catalytically inactive SENP1 bound to SUMO-1-modified RanGAP1 and to unprocessed SUMO-1. In each case, the scissile peptide bond is kinked at a right angle to the C-terminal tail of SUMO-1 and has the cis configuration of the amide nitrogens. SENP1 preferentially processes SUMO-1 over SUMO-2, but binding thermodynamics of full-length SUMO-1 and SUMO-2 to SENP1 and K(m) values for processing are very similar. However, k(cat) values differ by 50-fold. Thus, discrimination between unprocessed SUMO-1 and SUMO-2 by SENP1 is based on a catalytic step rather than substrate binding and is likely to reflect differences in the ability of SENP1 to correctly orientate the scissile bonds in SUMO-1 and SUMO-2.
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Endopeptidasas/química , Endopeptidasas/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Catálisis , Cristalografía por Rayos X , Cisteína/genética , Cisteína/metabolismo , Cisteína Endopeptidasas , Endopeptidasas/genética , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Proteína SUMO-1/genética , Especificidad por Sustrato , TermodinámicaRESUMEN
Introduction: Fluoroscopy-guided endourology procedures require proper radiation protection to minimize radiation risk. This multicenter study aimed at investigating radiation protection practice and related radiation exposure of operating team members. Materials and Methods: Six endourology centers from the South-Eastern European Group for Urolithiasis Research answered questionnaires and collected data of 315 procedures performed within a 3-months period, with simultaneous measurement of dose to staff and dose area product (DAP) to patient. A pair of calibrated personal dosimeters, one for body and one for eye-lens dose, was worn by all key staff members. Dosimeters were centrally calibrated, measured, and analyzed. Results: The annual workload ranged from 173 to 865 procedures per center. Practice of personal dose monitoring and use of radiation protection shielding was found to be inconsistent. Lead aprons and thyroid collars were used by all, whereas protective eyewear was used in only half of centers. Due to the regular use of protective aprons, the whole-body dose of all 44 monitored staff members was safely below the regulatory dose limits. Eye-lens dose of 17 (14 urologists and 3 assisting staff) was above the dosimeter detection level, and dose per procedure varied from <10 to 63 µSv. The highest annual eye-lens dose of 13.5 mSv was found for the surgeon in the busiest department by using an over-the-couch X-ray tube without a ceiling suspended screen. Working closer to patient body with no protection resulted in a six-time higher eye-lens dose per DAP for a surgeon compared with others in the same center. Lower eye-dose per procedure was associated with lower DAP to patient and with the use of an under-the-couch tube, lower fluoroscopy pulse rate, collimation, fluoroscopy time, and acquired images. Conclusions: The study results call for the need to establish standard protocols about use of fluoroscopy during endourology procedures and to increase radiation protection knowledge and awareness of surgical staff.
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Energía Nuclear , Exposición Profesional , Exposición a la Radiación , Urolitiasis , Fluoroscopía/efectos adversos , Humanos , Dosis de Radiación , Urolitiasis/cirugíaRESUMEN
Noninvasive detection of nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, promises to improve patient screening, accelerate drug trials, and reduce health care costs. On the basis of protease dysregulation of the biological pathways of fibrotic NASH, we developed the Glympse Bio Test System (GBTS) for multiplexed quantification of liver protease activity. GBTS-NASH comprises a mixture of 19 mass-barcoded PEGylated peptides that is administered intravenously and senses liver protease activity by releasing mass-barcoded reporters into urine for analysis by mass spectrometry. To identify a protease signature of NASH, transcriptomic analysis of 355 human liver biopsies identified a 13-protease panel that discriminated clinically relevant NASH ≥F2 fibrosis from F0-F1 with high classification accuracy across two independent patient datasets. We screened 159 candidate substrates to identify a panel of 19 peptides that exhibited high activity for our 13-protease panel. In the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) mouse model, binary classifiers trained on urine samples discriminated fibrotic NASH from simple steatosis and healthy controls across a range of nondisease conditions and indicated disease regression upon diet change [area under receiver operating characteristics (AUROCs) > 0.97]. Using a hepatoprotective triple combination treatment (FXR agonist, ACC and ASK1 inhibitors) in a rat model of NASH, urinary classification distinguished F0-F1 from ≥F2 animals and indicated therapeutic response as early as 1 week on treatment (AUROCs >0.91). Our results support GBTS-NASH to diagnose fibrotic NASH via an infusion of peptides, monitor changes in disease severity, and indicate early treatment response.
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Enfermedad del Hígado Graso no Alcohólico , Fibrosis , Humanos , PéptidosRESUMEN
Somites are embryonic precursors of the ribs, vertebrae and certain dermis tissue. Somite formation is a periodic process regulated by a molecular clock which drives cyclic expression of a number of clock genes in the presomitic mesoderm. To date the mechanism regulating the period of clock gene oscillations is unknown. Here we show that chick homologues of the Wnt pathway genes that oscillate in mouse do not cycle across the chick presomitic mesoderm. Strikingly we find that modifying Wnt signalling changes the period of Notch driven oscillations in both mouse and chick but these oscillations continue. We propose that the Wnt pathway is a conserved mechanism that is involved in regulating the period of cyclic gene oscillations in the presomitic mesoderm.
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Relojes Biológicos , Tipificación del Cuerpo/fisiología , Proteínas Wnt/metabolismo , Animales , Embrión de Pollo , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Ratones , Receptores Notch/metabolismo , Transducción de Señal , Somitos/metabolismo , Proteínas Wnt/genéticaRESUMEN
BACKGROUND: Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice. METHODS: Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying ß-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination. RESULTS: Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration. CONCLUSION: Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.
RESUMEN
Genome-wide association studies have implicated the TAM receptor tyrosine kinase (RTK) Mer in liver disease, yet our understanding of the role that Mer and its related RTKs Tyro3 and Axl play in liver homeostasis and the response to acute injury is limited. We find that Mer and Axl are most prominently expressed in hepatic Kupffer and endothelial cells and that as mice lacking these RTKs age, they develop profound liver disease characterized by apoptotic cell accumulation and immune activation. We further find that Mer is critical to the phagocytosis of apoptotic hepatocytes generated in settings of acute hepatic injury, and that Mer and Axl act in concert to inhibit cytokine production in these settings. In contrast, we find that Axl is uniquely important in mitigating liver damage during acetaminophen intoxication. Although Mer and Axl are protective in acute injury models, we find that Axl exacerbates fibrosis in a model of chronic injury. These divergent effects have important implications for the design and implementation of TAM-directed therapeutics that might target these RTKs in the liver.
Asunto(s)
Hígado/lesiones , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina Quinasa c-Mer/metabolismo , Animales , Apoptosis/genética , Células Endoteliales/metabolismo , Femenino , Estudio de Asociación del Genoma Completo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fagocitosis/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal/genética , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa del Receptor AxlRESUMEN
Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal-regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.
Asunto(s)
Muerte Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Cirrosis Hepática/metabolismo , Hígado/lesiones , Hígado/metabolismo , MAP Quinasa Quinasa Quinasa 5/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Inhibidores Enzimáticos/administración & dosificación , Femenino , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cirrosis Hepática/patología , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fosforilación , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.