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BACKGROUND AND OBJECTIVE: As the presentation of pulmonary nodules increases, the importance of a safe and accurate method of sampling peripheral pulmonary nodules is highlighted. First-generation robotic bronchoscopy has successfully assisted navigation and improved peripheral reach during bronchoscopy. Integrating tool-in-lesion tomosynthesis (TiLT) may further improve yield. METHODS: We performed a first-in-human clinical trial of a new robotic electromagnetic navigation bronchoscopy system with integrated digital tomosynthesis technology (Galaxy System, Noah Medical). Patients with moderate-risk peripheral pulmonary nodules were enrolled in the study. Robotic bronchoscopy was performed using electromagnetic navigation with TiLT-assisted lesion guidance. Non-specific results were followed up until either a clear diagnosis was achieved or repeat radiology at 6 months demonstrated stability. RESULTS: Eighteen patients (19 nodules) were enrolled. The average lesion size was 20 mm, and the average distance from the pleura was 11.6 mm. The target was successfully reached in 100% of nodules, and the biopsy tool was visualized inside the target lesion in all cases. A confirmed specific diagnosis was achieved in 17 nodules, 13 of which were malignant. In one patient, radiological monitoring confirmed a true non-malignant result. This translates to a yield of 89.5% (strict) to 94.7% (intermediate). Complications included one pneumothorax requiring observation only and another requiring an overnight chest drain. There was one case of severe pneumonia following the procedure. CONCLUSION: In this first-in-human study, second-generation robotic bronchoscopy using electromagnetic navigation combined with integrated digital tomosynthesis was feasible with an acceptable safety profile and demonstrated a high diagnostic yield for small peripheral lung nodules.
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The first dedicated tracheobronchial silicone stent was designed by the French pulmonologist Jean-Paul Dumon. The most common indications for stenting are to minimise extrinsic airway compression from mass effect, maintain airway patency due to intrinsic obstruction or treat significant nonmalignant airway narrowing or fistulae. Silicone stents require rigid bronchoscopy for insertion; however, they are more readily repositioned and removed compared with metallic stents. Metallic stents demonstrate luminal narrowing when loads are applied to their ends, therefore stents should either be reinforced at the ends or exceed the area of stenosis by a minimum of 5 mm. Nitinol, a nickel-titanium metal alloy, is currently the preferred material used for airway stents. Airway stenting provides effective palliation for patients with severe symptomatic obstruction. Drug-eluting and three-dimensional printing of airway stents present promising solutions to the challenges of the physical and anatomical constraints of the tracheobronchial tree. Biodegradable stents could also be a solution for the treatment of nonmalignant airway obstruction.
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Obstrucción de las Vías Aéreas , Broncoscopía , Níquel , Titanio , Humanos , Broncoscopía/métodos , Obstrucción de las Vías Aéreas/cirugía , Siliconas , Metales , Stents , Resultado del TratamientoRESUMEN
In the presence of polyvalent cations, long double-stranded DNA (dsDNA) in dilute solution undergoes a single-molecule, first-order, phase transition ("condensation"), a phenomenon that has been documented and analyzed by many years of experimental and theoretical studies. There has been no systematic effort, however, to determine whether long single-stranded RNA (ssRNA) shows an analogous behavior. In this study, using dynamic light scattering, analytical ultracentrifugation, and gel electrophoresis, we examine the effects of increasing polyvalent cation concentrations on the effective size of long ssRNAs ranging from 3000 to 12,000 nucleotides. Our results indicate that ssRNA does not undergo a discontinuous condensation as does dsDNA but rather a "continuous" decrease in size with increasing polyvalent cation concentration. And, instead of the 10-fold decrease in size shown by long dsDNA, we document a 50% decrease, as demonstrated for a range of lengths and sequences of ssRNA.
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ADN , ARN , ARN/genética , CationesRESUMEN
Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2) - which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin.Communicated by Ramaswamy H. Sarma.
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Actinas , Proteínas de Microfilamentos , Actinas/química , Forminas/metabolismo , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismoRESUMEN
Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and ß-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.
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Proteínas Bacterianas , Glicosiltransferasas , Lipoproteínas , Staphylococcus aureus , Ácidos Teicoicos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Staphylococcus aureus/metabolismo , Especificidad por Sustrato , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Uridina Difosfato/metabolismoRESUMEN
Endoscopic lung volume reduction (ELVR) is recognised in both national and international expert guidelines as one of the few additive treatments to benefit patients with advanced chronic obstructive pulmonary disease (COPD) who are otherwise receiving optimal medical and supportive care. Despite these recommendations and a growing evidence base, these procedures are not widely offered across Australia and New Zealand, and general practitioner and physician awareness of this therapy can be improved. ELVR aims to mitigate the impact of hyperinflation and gas trapping on dyspnoea and exercise intolerance in COPD. Effective ELVR is of proven benefit in improving symptoms, quality of life, lung function and survival. Several endoscopic techniques to achieve ELVR have been developed, with endobronchial valve placement to collapse a single lobe being the most widely studied and commonly practised. This review describes the physiological rationale underpinning lung volume reduction, highlights the challenges of patient selection, and provides an overview of the evidence for current and investigational endoscopic interventions for COPD.
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Broncoscopía/métodos , Disnea/fisiopatología , Neumonectomía/instrumentación , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/cirugía , Australia/epidemiología , Concienciación , Broncoscopía/normas , Humanos , Nueva Zelanda/epidemiología , Selección de Paciente/ética , Neumonectomía/métodos , Neumonectomía/mortalidad , Guías de Práctica Clínica como Asunto , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Calidad de Vida , Ensayos Clínicos Controlados Aleatorios como Asunto , Volumen Residual/fisiología , Instrumentos Quirúrgicos/efectos adversos , Sobrevida , Capacidad Pulmonar Total/fisiologíaRESUMEN
Rationale: Transbronchial lung cryobiopsy (TBLC) is an emerging technique for interstitial lung disease diagnosis. Good histopathologic agreement between TBLC and surgical lung biopsy (SLB) was demonstrated in the COLDICE (Cryobiopsy versus Open Lung Biopsy in the Diagnosis of Interstitial Lung Disease Alliance) study; however, diagnostic confidence was frequently lower for TBLC than SLB. Objectives: To characterize specific features of TBLC predictive of usual interstitial pneumonia (UIP) in corresponding SLB and to identify clinical indices predictive of biopsy concordance. Methods: The COLDICE study was a prospective, multicenter study investigating diagnostic agreement between TBLC and SLB. The participants underwent both procedures with blinded pathologist analysis of specimens, applying international guideline criteria. The TBLC features predictive of UIP in the paired SLB and predictive features of overall concordance were analyzed. Measurements and Main Results: A total of 65 patients (66.1 ± 9.3 yr; FVC, 84.7 ± 14.2%; DlCO, 63.4 ± 13.8%) participated in the COLDICE study. UIP was identified in 33/65 (50.8%) SLB, and 81.5% were concordant with corresponding TBLC (κ, 0.61; 95% confidence interval [CI], 0.38-0.77). The UIP guideline criteria of "predominantly subpleural or paraseptal fibrosis" was infrequently reported in TBLC (8/33, 24.2%), whereas "patchy fibrosis," "fibroblast foci," and the "absence of alternative diagnostic features" were frequently observed in TBLC. The combination of these three features strongly predicted UIP in paired SLB (odds ratio [OR], 23.4; 95% CI, 6.36-86.1; P < 0.0001). Increased numbers of TBLC samples predicted histopathologic concordance with SLB (OR, 1.8; 95% CI, 1.08-3.01; P = 0.03). The predictors of discordance included older age, family history, and radiologic asymmetry. Conclusions: Subpleural and/or paraseptal fibrosis were not essential for diagnosing UIP in TBLC, provided that other guideline criteria features were present. The diagnostic accuracy of TBLC was strengthened when increased numbers of samples were taken. Clinical trial registered with www.anzctr.org.au (ACTRN12615000718549).
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Biopsia , Broncoscopía , Criocirugía , Fibrosis Pulmonar Idiopática/patología , Anciano , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
Chemical risk assessment relies on toxicity tests that require significant numbers of animals, time and costs. For the >30,000 chemicals in commerce, the current scale of animal testing is insufficient to address chemical safety concerns as regulatory and product stewardship considerations evolve to require more comprehensive understanding of potential biological effects, conditions of use, and associated exposures. We demonstrate the use of a multi-level new approach methodology (NAMs) strategy for hazard- and risk-based prioritization to reduce animal testing. A Level 1/2 chemical prioritization based on estrogen receptor (ER) activity and metabolic activation using ToxCast data was used to select 112 chemicals for testing in a Level 3 human uterine cell estrogen response assay (IKA assay). The Level 3 data were coupled with quantitative in vitro to in vivo extrapolation (Q-IVIVE) to support bioactivity determination (as a surrogate for hazard) in a tissue-specific context. Assay AC50s and Q-IVIVE were used to estimate human equivalent doses (HEDs), and HEDs were compared to rodent uterotrophic assay in vivo-derived points of departure (PODs). For substances active both in vitro and in vivo, IKA assay-derived HEDs were lower or equivalent to in vivo PODs for 19/23 compounds (83%). Activity exposure relationships were calculated, and the IKA assay was as or more protective of human health than the rodent uterotrophic assay for all IKA-positive compounds. This study demonstrates the utility of biologically relevant fit-for-purpose assays and supports the use of a multi-level strategy for chemical risk assessment.
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Alternativas al Uso de Animales/métodos , Disruptores Endocrinos/toxicidad , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Toxicidad/métodos , Útero/efectos de los fármacos , Animales , Bioensayo/métodos , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Estudios de Factibilidad , Femenino , Humanos , Modelos Biológicos , Ratas , Medición de Riesgo/métodos , Útero/citologíaRESUMEN
BACKGROUND: Transbronchial lung cryobiopsy (TBLC) is a novel technique for sampling lung tissue for interstitial lung disease diagnosis. The aim of this study was to establish the diagnostic accuracy of TBLC compared with surgical lung biopsy (SLB), in the context of increasing use of TBLC in clinical practice as a less invasive biopsy technique. METHODS: COLDICE was a prospective, multicentre, diagnostic accuracy study investigating diagnostic agreement between TBLC and SLB, across nine Australian tertiary hospitals. Patients with interstitial lung disease aged between 18 and 80 years were eligible for inclusion if they required histopathological evaluation to aid diagnosis, after detailed baseline evaluation. After screening at a centralised multidisciplinary discussion (MDD), patients with interstitial lung disease referred for lung biopsy underwent sequential TBLC and SLB under one anaesthetic. Each tissue sample was assigned a number between 1 and 130, allocated in a computer-generated random sequence. Encoded biopsy samples were then analysed by masked pathologists. At subsequent MDD, de-identified cases were discussed twice with either TBLC or SLB along with clinical and radiological data, in random non-consecutive order. Co-primary endpoints were agreement of histopathological features in TBLC and SLB for patterns of definite or probable usual interstitial pneumonia, indeterminate for usual interstitial pneumonia, and alternative diagnosis; and for agreement of consensus clinical diagnosis using TBLC and SLB at MDD. Concordance and κ values were calculated for each primary endpoint. This study is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12615000718549. FINDINGS: Between March 15, 2016, and April 15, 2019, we enrolled 65 patients (31 [48%] men, 34 [52%] women; mean age 66·1 years [SD 9·3]; forced vital capacity 83·7% [SD 14·2]; diffusing capacity for carbon monoxide 63·4% [SD 12·8]). TBLC (7·1 mm, SD 1·9) and SLB (46·5 mm, 14·9) samples were each taken from two separate ipsilateral lobes. Histopathological agreement between TBLC and SLB was 70·8% (weighted κ 0·70, 95% CI 0·55-0·86); diagnostic agreement at MDD was 76·9% (κ 0·62, 0·47-0·78). For TBLC with high or definite diagnostic confidence at MDD (39 [60%] of 65 cases), 37 (95%) were concordant with SLB diagnoses. In the 26 (40%) of 65 cases with low-confidence or unclassifiable TBLC diagnoses, SLB reclassified six (23%) to alternative high-confidence or definite MDD diagnoses. Mild-moderate airway bleeding occurred in 14 (22%) patients due to TBLC. The 90-day mortality was 2% (one of 65 patients), following acute exacerbation of idiopathic pulmonary fibrosis. INTERPRETATION: High levels of agreement between TBLC and SLB for both histopathological interpretation and MDD diagnoses were shown. The TBLC MDD diagnoses made with high confidence were particularly reliable, showing excellent concordance with SLB MDD diagnoses. These data support the clinical utility of TBLC in interstitial lung disease diagnostic algorithms. Further studies investigating the safety profile of TBLC are needed. FUNDING: University of Sydney, Hunter Medical Research Institute, Erbe Elektromedizin, Medtronic, Cook Medical, Rymed, Karl-Storz, Zeiss, and Olympus.
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Biopsia/estadística & datos numéricos , Broncoscopía/métodos , Criobiología/métodos , Fibrosis Pulmonar Idiopática/diagnóstico , Enfermedades Pulmonares Intersticiales/diagnóstico , Australia , Biopsia/métodos , Femenino , Humanos , Pulmón/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Capacidad VitalRESUMEN
Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.
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Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Isoformas de Proteínas/metabolismo , Factor de Células Madre/metabolismo , Animales , Bleomicina/farmacología , Recuento de Células/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Pulmón/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In the past 10 years, the public, private, and non-profit sectors have found agreement that hazard identification and risk assessment should capitalize on the explosion of knowledge in the biological sciences, moving away from in life animal testing toward more human-relevant in vitro and in silico methods, collectively referred to as new approach methodologies (NAMs). The goals for implementation of NAMs are to efficiently identify possible chemical hazards and to gather dose-response data to inform more human-relevant safety assessment. While work proceeds to develop NAMs, there has been less emphasis on creating decision criteria or showing how risk context should guide selection and use of NAMs. Here, we outline application scenarios for NAMs in different risk contexts and place different NAMs and conventional testing approaches into four broad levels. Level 1 relies solely on computational screening; Level 2 consists of high throughput in vitro screening with human cells intended to provide broad coverage of possible responses; Level 3 focuses on fit-for-purpose assays selected based on presumptive modes of action (MOA) and designed to provide more quantitative estimates of relevant dose responses; Level 4 has a variety of more complex multi-dimensional or multi-cellular assays and might include targeted in vivo studies to further define MOA. Each level also includes decision-appropriate exposure assessment tools. Our aims here are to (1) foster discussion about context-dependent applications of NAMs in relation to risk assessment needs and (2) describe a functional roadmap to identify where NAMs are expected to be adequate for chemical safety decision-making.
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Alternativas a las Pruebas en Animales/tendencias , Pruebas de Toxicidad/tendencias , Animales , Biología Computacional/métodos , Química Computacional/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro , MamíferosRESUMEN
BACKGROUND: Endobronchial ultrasound-guided trans-bronchial needle aspiration (EBUS-TBNA) is minimally invasive technique used for diagnosis and/or staging of benign and malignant pulmonary and non-pulmonary disease. Previous studies have established the utility of EBUS-TBNA in narrowly defined indications and populations. In this pragmatic 'real world' study we have analysed the use of EBUS-TBNA for a variety of clinical presentations and its clinical application in conjunction with other invasive investigations. METHODS: All EBUS-TBNA procedures performed at Sir Charles Gardiner Hospital in 2012-2014 were reviewed retrospectively, using relevant hospital databases. RESULTS: A total of 327 patients underwent 337 EBUS-TBNA procedures. EBUS-TBNA procedures were used to diagnose a wide spectrum of benign and malignant conditions. The main application was in the diagnosis and staging of malignant conditions (70.6%), and in the diagnosis of benign conditions such as sarcoidosis 40 (12.2%), and silicoanthracosis 17 (5.2%). EBUS-TBNA was sufficient to diagnose and stage the disease as a single stand-alone invasive procedure in 191 (59.2%) patients. EBUS-TBNA was the final invasive procedure undertaken in 283 (87.6%) patients. Only 13.3% of non small cell lung cancer (NSCLC) patients who had EBUS-TBNA as a first investigation required multiple procedures compared to 51.1% of all NSCLC patients undergoing EBUS-TBNA. Overall sensitivity, specificity, NPV and diagnostic accuracy for EBUS-TBNA were 89.7, 100, 85.1 and 89.9%, respectively and three minor complications (0.9%) occurred as a result of the procedure. CONCLUSIONS: EBUS-TBNA was undertaken for a wide variety of clinical conditions. Good diagnostic accuracy and safety profiles were demonstrated for the procedure, supporting its application as a first line investigation in the diagnosis and/or staging of a range of malignant and benign conditions. Our study was unique in its documentation of the use of EBUS-TBNA in a real-world setting in conjunction with other invasive modalities. EBUS-TBNA was utilised as a stand alone invasive procedure in more than half of the patients. Importantly, in NSCLC, when EBUS-TBNA was performed as primary diagnostic and staging investigation, less patients underwent subsequent invasive procedures.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico/estadística & datos numéricos , Enfermedades Pulmonares/patología , Neoplasias Pulmonares/patología , Sarcoidosis/patología , Anciano , Australia , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Bases de Datos Factuales , Femenino , Humanos , Enfermedades Pulmonares/diagnóstico , Neoplasias Pulmonares/diagnóstico , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Sarcoidosis/diagnóstico , Sensibilidad y Especificidad , Silicosis/diagnóstico , Silicosis/patologíaRESUMEN
Stem cell factor (SCF) binds to the receptor c-Kit that is expressed on a number of myeloid and lymphoid cell populations, including Type 2 innate lymphoid cells (ILC2). However the importance of the SCF/c-Kit interaction in ILC2 has not been studied. Here we investigate the role of a specific SCF isoform, SCF248, in the allergic asthmatic response and SCF/c-Kit in ILC2 activation during chronic allergy. We observed that mice treated with a monoclonal antibody specific for SCF248 attenuated the development of chronic asthmatic disease by decreasing the number of mast cells, ILC2 and eosinophils, as well as reducing the accompanying pathogenic cytokine responses. These data were supported using SCFfl/fl-Col1-Cre-ERT mice and W/Wv mice that demonstrated the importance of the stem cell factor/c-Kit activation during chronic allergy and the accumulation of c-kit+ cells. Finally, these data demonstrate for the first time that SCF could activate ILC2 cells in vitro for the production of key allergic cytokines. Together these findings indicate that SCF is a critical cytokine involved in the activation of ILC2 that lead to more severe outcomes during chronic allergy and that the SCF248 isoform could be an important therapeutic target to control the disease progression.
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Asma/inmunología , Pulmón/patología , Linfocitos/inmunología , Factor de Células Madre/metabolismo , Alérgenos/inmunología , Animales , Células Cultivadas , Enfermedad Crónica , Colágeno Tipo I/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/genética , Factor de Células Madre/inmunología , Células Th2/inmunologíaRESUMEN
In order to proliferate and mount an infection, many bacterial pathogens need to acquire iron from their host. The most abundant iron source in the body is the oxygen transporter hemoglobin (Hb). Streptococcus pyogenes, a potentially lethal human pathogen, uses the Shr protein to capture Hb on the cell surface. Shr is an important virulence factor, yet the mechanism by which it captures Hb and acquires its heme is not well-understood. Here, we show using NMR and biochemical methods that Shr binds Hb using two related modules that were previously defined as domains of unknown function (DUF1533). These hemoglobin-interacting domains (HIDs), called HID1 and HID2, are autonomously folded and independently bind Hb. The 1.5 Å resolution crystal structure of HID2 revealed that it is a structurally unique Hb-binding domain. Mutagenesis studies revealed a conserved tyrosine in both HIDs that is essential for Hb binding. Our biochemical studies indicate that HID2 binds Hb with higher affinity than HID1 and that the Hb tetramer is engaged by two Shr receptors. NMR studies reveal the presence of a third autonomously folded domain between HID2 and a heme-binding NEAT1 domain, suggesting that this linker domain may position NEAT1 near Hb for heme capture.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hemoglobinas/metabolismo , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Proteínas Bacterianas/genética , Hemo/metabolismo , Hemoglobinas/química , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Dominios Proteicos , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/química , Streptococcus pyogenes/genéticaRESUMEN
The epistemologies and politics of comparative research are prominently debated within urban studies, with 'comparative urbanism' emerging as a contemporary lexicon of urban studies. The study of urban gentrification has, after some delay, come to engage with these debates, which can be seen to pose a major challenge to the very concept of gentrification. To date, similar debates or developments have not unfolded within the study of rural gentrification. This article seeks to address some of the challenges posed to gentrification studies through an examination of strategies of comparison and how they might be employed within a comparative study of rural gentrification. Drawing on Tilly (Big structures Large Processes Huge Comparisons. New York: Russell Sage), examples of four 'strategies of comparison' are identified within studies of urban and rural gentrification, before the paper explores how 'geographies of the concept' and 'geographies of the phenomenon' of rural gentrification in the United Kingdom, United States and France may be investigated using Latour's (Pandora's Hope. London: Harvard University Press) notion of 'circulatory sociologies of translation'. The aim of our comparative discussion is to open up dialogues on the challenges of comparative studies that employ conceptions of gentrification and also to promote reflections of the metrocentricity of recent discussions of comparative research.
RESUMEN
In response to the five commentaries on our paper 'Comparative approaches to gentrification: lessons from the rural', we open up more 'windows' on rural gentrification and its urban counterpart. First, we highlight the issues of metrocentricity and urbanormativity within gentrification studies, highlighting their employment by our commentators. Second, we consider the issue of displacement and its operation within rural space, as well as gentrification as a coping strategy for neoliberal existence and connections to more-than-human natures. Finally, we consider questions of scale, highlighting the need to avoid naturalistic conceptions of scale and arguing that attention could be paid to the role of material practices, symbolizations and lived experiences in producing scaled geographies of rural and urban gentrification.
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Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It actively acquires the essential nutrient iron from human hemoglobin (Hb) using the iron-regulated surface-determinant (Isd) system. This process is initiated when the closely related bacterial IsdB and IsdH receptors bind to Hb and extract its hemin through a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. Previously, we demonstrated that the tri-domain unit within IsdH (IsdHN2N3) triggers hemin release by distorting Hb's F-helix. Here, we report that IsdHN2N3 promotes hemin release from both the α- and ß-subunits. Using a receptor mutant that only binds to the α-subunit of Hb and a stopped-flow transfer assay, we determined the energetics and micro-rate constants of hemin extraction from tetrameric Hb. We found that at 37 °C, the receptor accelerates hemin release from Hb up to 13,400-fold, with an activation enthalpy of 19.5 ± 1.1 kcal/mol. We propose that hemin removal requires the rate-limiting hydrolytic cleavage of the axial HisF8 Nϵ-Fe3+ bond, which, based on molecular dynamics simulations, may be facilitated by receptor-induced bond hydration. Isothermal titration calorimetry experiments revealed that two distinct IsdHN2N3·Hb protein·protein interfaces promote hemin release. A high-affinity receptor·Hb(A-helix) interface contributed â¼95% of the total binding standard free energy, enabling much weaker receptor interactions with Hb's F-helix that distort its hemin pocket and cause unfavorable changes in the binding enthalpy. We present a model indicating that receptor-introduced structural distortions and increased solvation underlie the IsdH-mediated hemin extraction mechanism.