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BACKGROUND: To say data is revolutionising the medical sector would be a vast understatement. The amount of medical data available today is unprecedented and has the potential to enable to date unseen forms of healthcare. To process this huge amount of data, an equally huge amount of computing power is required, which cannot be provided by regular desktop computers. These areas can be (and already are) supported by High-Performance-Computing (HPC), High-Performance Data Analytics (HPDA), and AI (together "HPC+"). OBJECTIVE: This overview article aims to show state-of-the-art examples of studies supported by the National Competence Centres (NCCs) in HPC+ within the EuroCC project, employing HPC, HPDA and AI for medical applications. METHOD: The included studies on different applications of HPC in the medical sector were sourced from the National Competence Centres in HPC and compiled into an overview article. Methods include the application of HPC+ for medical image processing, high-performance medical and pharmaceutical data analytics, an application for pediatric dosimetry, and a cloud-based HPC platform to support systemic pulmonary shunting procedures. RESULTS: This article showcases state-of-the-art applications and large-scale data analytics in the medical sector employing HPC+ within surgery, medical image processing in diagnostics, nutritional support of patients in hospitals, treating congenital heart diseases in children, and within basic research. CONCLUSION: HPC+ support scientific fields from research to industrial applications in the medical area, enabling researchers to run faster and more complex calculations, simulations and data analyses for the direct benefit of patients, doctors, clinicians and as an accelerator for medical research.
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Metodologías Computacionales , Programas Informáticos , Niño , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition in vitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and in vivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.
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Fibrosis Pulmonar Idiopática , Surfactantes Pulmonares , Humanos , Ratones , Animales , Tensoactivos , Pulmón , Células Epiteliales Alveolares , Bleomicina , Receptor Notch1RESUMEN
Fine-tuned regulation of protein biosynthesis is crucial for cellular fitness and became even more vital when cellular and organismal complexity increased during the course of evolution. In order to cope with this augmented demand for translation control, eukaryal ribosomes have gained extensions both at the ribosomal protein and rRNA levels. Here we analyze the functional role of ES27L, an rRNA expansion segment in the large ribosomal subunit of Saccharomyces cerevisiae. Deletion of the b-arm of this expansion segment, called ES27Lb, did not hamper growth during optimal conditions, thus demonstrating that this 25S rRNA segment is not inherently crucial for ribosome functioning. However, reductive stress results in retarded growth and rendered unique protein sets prone to aggregation. Lack of ES27Lb negatively affects ribosome-association of known co-translational N-terminal processing enzymes which in turn contributes to the observed protein aggregation. Likely as a compensatory response to these challenges, the truncated ribosomes showed re-adjusted translation of specific sets of mRNAs and thus fine-tune the translatome in order to re-establish proteostasis. Our study gives comprehensive insight into how a highly conserved eukaryal rRNA expansion segment defines ribosomal integrity, co-translational protein maturation events and consequently cellular fitness.
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Proteoma/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Unión Proteica/genética , ARN de Hongos/genética , ARN Ribosómico/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Duchenne muscular dystrophy is the most frequent lethal genetic disease. Several clinical trials have established both the beneficial effect of steroids in Duchenne muscular dystrophy and the well-known risk of side effects associated with their daily use. For many years it has been known that steroids associated with ambulation loss lead to obesity and also damage the bone structure resulting in the bone density reduction and increased incidence of bone fractures and fat embolism syndrome, an underdiagnosed complication after fractures. Fat embolism syndrome is characterized by consciousness disturbance, respiratory failure and skin rashes. The use of steroids in Duchenne muscular dystrophy may result in vertebral fractures, even without previous trauma. Approximately 25% of patients with Duchenne muscular dystrophy have a long bone fracture, and 1% to 22% of fractures have a chance to develop fat embolism syndrome. As the patients with Duchenne muscular dystrophy have progressive cardiac and respiratory muscle dysfunction, the fat embolism may be unnoticed clinically and may result in increased risk of death and major complications. Different treatments and prevention measures of fat embolism have been proposed; however, so far, there is no efficient therapy. The prevention, early diagnosis and adequate symptomatic treatment are of paramount importance. The fat embolism syndrome should always be considered in patients with Duchenne muscular dystrophy presenting with fractures, or an unexplained and sudden worsening of respiratory and cardiac symptoms.
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The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson-Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation.
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Antibacterianos/química , Macrólidos/química , ARN Ribosómico 23S/química , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Bacteriano/química , ThermusRESUMEN
Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1ß and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1ß, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.
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Células Presentadoras de Antígenos/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Citocinas/metabolismo , Deficiencia del Factor XII/metabolismo , Factor XII/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/inmunología , Proliferación Celular , Citocinas/inmunología , Modelos Animales de Enfermedad , Factor XII/genética , Deficiencia del Factor XII/sangre , Deficiencia del Factor XII/genética , Deficiencia del Factor XII/inmunología , Factor XIIa/genética , Factor XIIa/metabolismo , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/inmunología , Activación de Linfocitos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Fenotipo , Placa Aterosclerótica , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Factores de TiempoRESUMEN
Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, ß-NAC and ß'-NAC) and of different NAC heterodimers found in yeast (αß-NAC and αß'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αß-NAC but not its paralogue αß'-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αß-NAC and αß'-NAC are functionally distinct from each other. The function of αß-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual ß-NAC, ß'-NAC or α-NAC subunits as well as αß'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only ß-NAC was able to restore growth defects suggesting chaperoning activities for ß-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αß-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.
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Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
OBJECTIVE: Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)-mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1α in APCs in atherosclerosis. APPROACH AND RESULTS: We found upregulated HIF1α expression in CD11c(+) APCs within atherosclerotic plaques of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Conditional deletion of Hif1a in CD11c(+) APCs in high-fat diet-fed Ldlr(-/-) mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1α directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1α-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr(-/-) mice. Notably, deletion of Hif1a in LysM(+) bone marrow cells in Ldlr(-/-) mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1α, STAT3, and interleukin-12 protein were found to colocalize with APCs. CONCLUSIONS: Our findings identify HIF1α to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr(-/-) mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.
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Células Presentadoras de Antígenos/metabolismo , Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-12/metabolismo , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.
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Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Homeostasis , Polilisina/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Proteínas de Unión a Caperuzas de ARN/genética , Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras , Ribonucleasas/genética , Ribonucleasas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.
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GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Oxígeno/metabolismo , Fosfatos/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Ribosómico/metabolismo , Catálisis , Histidina/metabolismo , Hidrólisis , Modelos Biológicos , Mutagénesis , Compuestos Organofosforados/metabolismo , Factor G de Elongación Peptídica/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Fosfatos/análisisRESUMEN
RATIONALE: Proinflammatory adaptive immune responses are recognized as major drivers of atherosclerotic lesion formation. Although CD8(+) T cells have recently been proposed as a proatherogenic cell subset, their full scope of actions remains to be elucidated. OBJECTIVE: We here addressed the contribution of CD8(+) T cells to monocyte trafficking in atherosclerosis. METHOD AND RESULTS: We observed that CD8(+) T cells express proinflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-12) within atherosclerotic lesions and spleens of high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Antibody-mediated CD8(+) T-cell depletion in high-fat diet-fed Ldlr(-/-) mice decreased atherosclerotic plaque formation, associated with decreased macrophage accumulation within lesions. Despite a reduction in vascular chemokine (CC-motif) ligand 2 and chemokine (CXC-motif) ligand 1 expression, CD8(+) T-cell depletion did not directly affect monocyte recruitment to inflamed vessels. However, CD8(+) T-cell depletion decreased chemokine (CC-motif) ligand serum concentrations and circulating Ly6C(high) monocyte counts. We further evidenced that CD8(+) T-cell depletion decreased levels of mature monocytes and myeloid granulocyte-monocyte progenitors in the bone marrow and spleen of hypercholesterolemic mice, effects that were partially reproduced by interferon-γ neutralization, showing a role for interferon-γ. CONCLUSIONS: These data suggest that CD8(+) T cells promote atherosclerosis by controlling monopoiesis and circulating monocyte levels, which ultimately contributes to plaque macrophage burden without affecting direct monocyte recruitment, identifying this cell subset as a critical regulator of proatherogenic innate immune cell responses in atherosclerosis.
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Antígenos Ly/análisis , Aterosclerosis/inmunología , Linfocitos T CD8-positivos/inmunología , Monocitos/inmunología , Mielopoyesis/inmunología , Animales , Suero Antilinfocítico/uso terapéutico , Aterosclerosis/etiología , Médula Ósea/metabolismo , Médula Ósea/patología , Linfocitos T CD8-positivos/metabolismo , Estenosis Carotídea/inmunología , Estenosis Carotídea/patología , Células Cultivadas , Quimiotaxis de Leucocito/inmunología , Citocinas/biosíntesis , Citocinas/genética , Citocinas/metabolismo , Dieta Aterogénica/efectos adversos , Grasas de la Dieta/toxicidad , Endarterectomía Carotidea , Regulación de la Expresión Génica/inmunología , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/etiología , Interferón gamma/fisiología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.
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Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Thermus/química , Thermus/citología , Emparejamiento Base , MutagénesisRESUMEN
Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall. As part of a tightly connected cross-talk between inflammation and coagulation, there is growing evidence that the coagulation system plays a pivotal role in the development and progression of atherosclerosis. We here discuss the presence of coagulation factors in atherosclerotic lesions and the overall effects of hypercoagulability and hypocoagulability on atherosclerotic lesion formation. Moreover, we focus on the unifying common pathway of coagulation, which can be initiated by the intrinsic and extrinsic pathway of coagulation, and discuss the functions of the coagulation factors FX, thrombin, and FXIII as regulators of inflammation in atherosclerosis. In particular, we review the non-hemostatic and immune-modulatory functions of these factors in endothelial and smooth muscle cells, as well as monocytes/macrophages, but also other cells, such as dendritic cells and T cells, that may control the inflammatory process of atherosclerosis. Their multiple roles in coagulation, but also their non-hemostatic functions in different cell types in inflammation and immunity, may harbor great potential for the development of novel therapeutic approaches for treating cardiovascular disease.
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Aterosclerosis/fisiopatología , Factores de Coagulación Sanguínea/metabolismo , Coagulación Sanguínea/fisiología , Pleiotropía Genética/fisiología , Inflamación/fisiopatología , Miocitos del Músculo Liso/metabolismo , Animales , Aterosclerosis/tratamiento farmacológico , Carbohidrato Epimerasas/metabolismo , Línea Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Factor XIII/metabolismo , Humanos , Cetona Oxidorreductasas/metabolismo , Ratones , Trombina/metabolismoRESUMEN
T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3(+) regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4(+) T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr(-/-)Pd1(-/-)) displayed striking increases in systemic CD4(+) and CD8(+) T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr(-/-)Pd1(-/-) mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr(-/-) mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.
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Aterosclerosis/genética , Aterosclerosis/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Receptor de Muerte Celular Programada 1/deficiencia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Aterosclerosis/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hipercolesterolemia/genética , Hipercolesterolemia/inmunología , Inmunomodulación/genética , Inmunomodulación/inmunología , Lípidos/sangre , Recuento de Linfocitos , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Masculino , Ratones , Ratones Noqueados , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Dendritic cells (DCs) can be sub-divided into various subsets that play specialized roles in priming of adaptive immune responses. Atherosclerosis is regarded as a chronic inflammatory disease of the vessel wall and DCs can be found in non-inflamed and diseased arteries. We here performed a systematic analyses of DCs subsets during atherogenesis. Our data indicate that distinct DC subsets can be localized in the vessel wall. In C57BL/6 and low density lipoprotein receptor-deficient (Ldlr (-/-)) mice, CD11c(+) MHCII(+) DCs could be discriminated into CD103(-) CD11b(+)F4/80(+), CD11b(+)F4/80(-) and CD11b(-)F4/80(-) DCs and CD103(+) CD11b(-)F4/80(-) DCs. Except for CD103(-) CD11b(-) F4/80(-) DCs, these subsets expanded in high fat diet-fed Ldlr (-/-) mice. Signal-regulatory protein (Sirp)-α was detected on aortic macrophages, CD11b(+) DCs, and partially on CD103(-) CD11b(-) F4/80(-) but not on CD103(+) DCs. Notably, in FMS-like tyrosine kinase 3-ligand-deficient (Flt3l (-/-)) mice, a specific loss of CD103(+) DCs but also CD103(-) CD11b(+) F4/80(-) DCs was evidenced. Aortic CD103(+) and CD11b(+) F4/80(-) CD103(-) DCs may thus belong to conventional rather than monocyte-derived DCs, given their dependence on Flt3L-signalling. CD64, postulated to distinguish macrophages from DCs, could not be detected on DC subsets under physiological conditions, but appeared in a fraction of CD103(-) CD11b(+) F4/80(-) and CD11b(+) F4/80(+) cells in atherosclerotic Ldlr (-/-) mice. The emergence of CD64 expression in atherosclerosis may indicate that CD11b(+) F4/80(-) DCs similar to CD11b(+) F4/80(+) DCs are at least in part derived from immigrated monocytes during atherosclerotic lesion formation. Our data advance our knowledge about the presence of distinct DC subsets and their accumulation characteristics in atherosclerosis, and may help to assist in future studies aiming at specific DC-based therapeutic strategies for the treatment of chronic vascular inflammation.
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Aorta/inmunología , Aorta/patología , Aterosclerosis/inmunología , Aterosclerosis/patología , Células Dendríticas/inmunología , Animales , Antígeno CD11c/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
The chemokine receptor CCR6 is expressed by various cell subsets implicated in atherogenesis, such as monocytes, Th17 and regulatory T cells. In order to further define the role of CCR6 in atherosclerosis, CCR6-deficient (Ccr6-/-) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr-/-) mice to generate atherosclerosis-prone mice deficient in CCR6. Compared to Ldlr-/- controls, atherosclerotic burden in the aortic sinus and aorta were reduced in Ccr6-/-Ldlr-/- mice fed a high fat diet, associated with a profound depression in lesional macrophage accumulation. Local and systemic distributions of T cells, including frequencies of Th1, Th17 and regulatory T cells were unaltered. In contrast, circulating counts of both Gr-1(high) and Gr1(low) monocytes were reduced in Ccr6-/-Ldlr-/- mice. Moreover, CCR6 was revealed to promote monocyte adhesion to inflamed endothelium in vitro and leukocyte adhesion to carotid arteries in vivo. Finally, CCR6 selectively recruited monocytes but not T cells in an acute inflammatory air pouch model. We here show that CCR6 functions on multiple levels and regulates the mobilisation, adhesion and recruitment of monocytes/macrophages to the inflamed vessel, thereby promoting atherosclerosis, but is dispensable for hypercholesterolaemia-associated adaptive immune priming. Targeting CCR6 or its ligand CCL20 may therefore be a promising therapeutic strategy to alleviate atherosclerosis.
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Aterosclerosis/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Receptores CCR6/metabolismo , Linfocitos T/inmunología , Animales , Adhesión Celular/genética , Movimiento Celular/genética , Células Cultivadas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Inflamación/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR6/genética , Receptores CCR6/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de LDL/genéticaRESUMEN
BACKGROUND: Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. RESULTS: For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. CONCLUSION: Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.
Asunto(s)
Clonación Molecular/métodos , ADN/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , ADN/genética , Electroforesis en Gel de Poliacrilamida , Ingeniería Genética , Histidina , Immunoblotting , Oligopéptidos , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1 , SolubilidadRESUMEN
Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.
Asunto(s)
Aterosclerosis/inmunología , Quimiocina CCL17/inmunología , Células Dendríticas/inmunología , Homeostasis , Linfocitos T Reguladores/inmunología , Animales , Trasplante de Médula Ósea , Movimiento Celular , Quimiocina CCL17/genética , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Shear forces are generated in all parts of the vascular system and contribute directly and indirectly to vascular disease progression. Endothelial cells are able to adapt to flow conditions, and are known to polarize and migrate in response to shear forces. Platelets exposed to shear stress are activated and release bioactive molecules from their alpha granules. So far, platelets have been considered to be static cells that do not leave the site of tight adhesion. However, we have recently been able to demonstrate the capacity of platelets to migrate in response to stromal derived factor-1 (SDF-1). In this project, we have demonstrated that platelets accumulate in areas with a high concentration of SDF-1 under flow conditions and respond to high shear stress by cellular polarization, cytoskeletal reorganisation, and flow-directed migration. In this context, we have shown increased Wiskott-Aldrich Syndrome protein (WASP) phosphorylation and intracellular redistribution of focal adhesion kinase (FAK) under high-shear stress conditions. The effect of flow-induced platelet migration has not previously been recognized and offers a new role for platelets as mobile cells. Their migratory potential may enable platelets to cover intimal lesions and contribute to vascular repair.
Asunto(s)
Plaquetas , Movimiento Celular , Quimiocina CXCL12/farmacología , Plaquetas/citología , Plaquetas/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Polaridad Celular , Quimiocina CXCL12/metabolismo , Endotelio Vascular/metabolismo , Citometría de Flujo , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Hemodinámica/efectos de los fármacos , Humanos , Microscopía Confocal , Fosforilación/efectos de los fármacos , Estrés Mecánico , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Platelets have been regarded as static cells that do not move once they adhere to a matrix. The present study explored, whether platelets are able to migrate. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrate through a transwell membrane and endothelium toward a source of stromal cell-derived factor 1 (SDF-1). Platelet migration was stimulated by SDF-1, which led to the downstream activation and phosphorylation of Wiskott-Aldrich syndrome protein. SDF-1 signaling and subsequent platelet migration could be inhibited by CXCR4-receptor blocker AMD3100, pertussis toxin, inhibition of phosphoinositol 3-kinase (PI3 kinase) with LY294002 or wortmannin, and disruption of actin polymerization with cytochalasin B. The potential of platelets to migrate in an SDF-1-mediated fashion may redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration, and vascular regeneration.