RESUMO
BACKGROUND: Pulmonary exacerbations (PExs) are clinically important in people with cystic fibrosis (CF). Multiple definitions have been used for PEx, and this scoping review aimed to identify the different definitions reported in the literature and to ascertain which signs and symptoms are commonly used to define them. METHODS: A search was performed using Embase, MEDLINE, Cochrane Library, Scopus and CINAHL. All publications reporting clinical trials or prospective observational studies involving definitions of PEx in people with CF published in English from January 1990 to December 2022 were included. Data were then extracted for qualitative thematic analysis. RESULTS: A total of 14 039 records were identified, with 7647 titles and abstracts screened once duplicates were removed, 898 reviewed as full text and 377 meeting the inclusion criteria. Pre-existing definitions were used in 148 publications. In 75% of papers, an objective definition was used, while 25% used a subjective definition, which subcategorised into treatment-based definitions (76%) and those involving clinician judgement (24%). Objective definitions were subcategorised into three groups: those based on a combination of signs and symptoms (50%), those based on a predefined combination of signs and symptoms plus the initiation of acute treatment (47%) and scores involving different clinical features each with a specific weighting (3%). The most common signs and symptoms reported in the definitions were, in order, sputum production, cough, lung function, weight/appetite, dyspnoea, chest X-ray changes, chest sounds, fever, fatigue or lethargy and haemoptysis. CONCLUSION: We have identified substantial variation in the definitions of PEx in people with CF reported in the literature. There is a requirement for the development of internationally agreed-upon, standardised and validated age-specific definitions. Such definitions would allow comparison between studies and effective meta-analysis to be performed and are especially important in the highly effective modulator therapy era in CF care.
Assuntos
Fibrose Cística , Progressão da Doença , Fibrose Cística/fisiopatologia , Fibrose Cística/complicações , HumanosRESUMO
Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air-liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Análise Espectral Raman , Humanos , Análise Espectral Raman/métodos , Concentração de Íons de Hidrogênio , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Compostos de Sulfidrila/química , Benzoatos/químicaRESUMO
Cystic fibrosis is a multiorgan disease caused by impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR). Since the introduction of the CFTR modulator combination elexacaftor-tezacaftor-ivacaftor (ETI), which acts directly on mutant CFTR to enhance its activity, most people with cystic fibrosis (pwCF) have seen pronounced reductions in symptoms, and studies project marked increases in life expectancy for pwCF who are eligible for ETI. However, modulator therapy has not cured cystic fibrosis and the success of CFTR modulators has resulted in immediate questions about the new state of cystic fibrosis disease and clinical challenges in the care of pwCF. In this Series paper, we summarise key questions about cystic fibrosis disease in the era of modulator therapy, highlighting state-of-the-art research and clinical practices, knowledge gaps, new challenges faced by pwCF and the potential for future health-care challenges, and the pressing need for additional therapies to treat the underlying genetic or molecular causes of cystic fibrosis.
Assuntos
Fibrose Cística , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Atenção à Saúde , Aminofenóis/uso terapêutico , Benzodioxóis/uso terapêutico , Mutação , Agonistas dos Canais de Cloreto/uso terapêuticoRESUMO
An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5' end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.
Assuntos
Fibrose Cística , Humanos , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Mutação , Pseudomonas aeruginosaRESUMO
Patient derived organoids have the potential to improve the physiological relevance of in vitro disease models. However, the 3D architecture of these self-assembled cellular structures makes probing their biochemistry more complex than in traditional 2D culture. We explore the application of surface enhanced Raman scattering microsensors (SERS-MS) to probe local pH gradients within patient derived airway organoid cultures. SERS-MS consist of solid polymer cores decorated with surface immobilised gold nanoparticles which are functionalised with pH sensitive reporter molecule 4-mercaptobenzoic acid (MBA). We demonstrate that by mixing SERS-MS into the extracellular matrix (ECM) of airway organoid cultures the probes can be engulfed by expanding organoids and report on local pH in the organoid lumen and ECM.
Assuntos
Ouro , Nanopartículas Metálicas , Humanos , Ouro/química , Nanopartículas Metálicas/química , Células-Tronco , Organoides/metabolismo , Matriz Extracelular , Análise Espectral Raman , Concentração de Íons de HidrogênioRESUMO
Rationale: The etiology of cystic fibrosis (CF) pulmonary exacerbations (PEx) is likely multifactorial with viral, bacterial, and non-infectious pathways contributing. Objectives: To determine whether viral infection status and CRP (C-reactive protein) can classify subphenotypes of PEx that differ in outcomes and biomarker profiles. Methods: Patients were recruited at time of admission for a PEx. Nasal swabs and sputum samples were collected and processed using the respiratory panel of the FilmArray multiplex polymerase chain reaction (PCR). Serum and plasma biomarkers were measured. PEx were classified using serum CRP and viral PCR: "pauci-inflammatory" if CRP < 5 mg/L, "non-viral with systemic inflammation" if CRP ⩾ 5 mg/L and no viral infection detected by PCR and "viral with systemic inflammation" if CRP ⩾ 5 mg/L and viral infection detected by PCR. Results: Discovery cohort (n = 59) subphenotype frequencies were 1) pauci-inflammatory (37%); 2) non-viral with systemic inflammation (41%); and 3) viral with systemic inflammation (22%). Immunoglobulin G, immunoglobulin M, interleukin-10, interleukin-13, serum calprotectin, and CRP levels differed across phenotypes. Reduction from baseline in forced expiratory volume in 1 second as percent predicted (FEV1pp) at onset of exacerbation differed between non-viral with systemic inflammation and viral with systemic inflammation (-6.73 ± 1.78 vs. -13.5 ± 2.32%; P = 0.025). Non-viral with systemic inflammation PEx had a trend toward longer duration of intravenous antibiotics versus pauci-inflammation (18.1 ± 1.17 vs. 14.8 ± 1.19 days, P = 0.057). There were no differences in percent with lung function recovery to <10% of baseline FEV1pp. Similar results were seen in local and external validation cohorts comparing a pauci-inflammatory to viral/non-viral inflammatory exacerbation phenotypes. Conclusions: Subphenotypes of CF PEx exist with differences in biomarker profile, clinical presentation, and outcomes.
Assuntos
Fibrose Cística , Humanos , Pulmão , Proteína C-Reativa/metabolismo , Antibacterianos/uso terapêutico , Biomarcadores , Inflamação/tratamento farmacológico , Fenótipo , Progressão da DoençaRESUMO
We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34-70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.
Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dicroísmo Circular , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Macrophages represent prominent immune orchestrators of cystic fibrosis (CF) inflammation and, as such, are an ever-increasing focus of CF research with several reports of intrinsic immune dysfunction related to loss of CFTR activity in macrophages themselves. Animal models of CF have contributed, in no small part, to a deepening of our understanding of the pathophysiology of the disease and towards therapeutic development. A commonly-used animal model in CF research is the Cftrtm1Unc Tg(FABP-hCFTR) mouse, which displays gut-specific expression of a human CFTR transgene in order to rescue the high rate of early mortality in Cftr-null mice associated with severe intestinal obstruction. We find significant variation in the response to inflammatory challenge of patient macrophages and cells derived from the Cftrtm1Unc Tg(FABP-hCFTR) mouse and show that macrophages derived from this mouse exhibit aberrant expression of human CFTR. This may contribute to the absence of inflammatory changes in this model.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Intestinos/metabolismo , Animais , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos CFTR , FenótipoRESUMO
Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4+ or CD8+ T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.
Assuntos
Separação Celular , Citometria de Fluxo , Neutrófilos/imunologia , Antígenos CD/metabolismo , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Microambiente Celular , Centrifugação com Gradiente de Concentração , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Armadilhas Extracelulares/metabolismo , Voluntários Saudáveis , Homeostase , Humanos , Interferon gama/metabolismo , Contagem de Leucócitos , Transtornos Leucocíticos/imunologia , Transtornos Leucocíticos/metabolismo , Ativação Linfocitária , Ativação de Neutrófilo , Neutrófilos/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 -DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.
Assuntos
Espectrometria de Fluorescência , Proteínas de Ligação a Telômeros/metabolismo , Temperatura , Quadruplex G , Humanos , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Complexo Shelterina , Telômero/química , Telômero/metabolismo , Proteínas de Ligação a Telômeros/químicaRESUMO
The Inflammatory Bowel Diseases (IBD), Ulcerative Colitis (UC) and Crohn's Disease (CD) are characterised by chronic non-resolving gut mucosal inflammation involving innate and adaptive immune responses. Neutrophils, usually regarded as first responders in inflammation, are a key presence in the gut mucosal inflammatory milieu in IBD. Here, we review the role of neutrophil extracellular trap (NET) formation as a potential effector disease mechanism. NETs are extracellular webs of chromatin, microbicidal proteins and oxidative enzymes that are released by neutrophils to contain pathogens. NETs contribute to the pathogenesis of several immune-mediated diseases such as systemic lupus erythematosus and rheumatoid arthritis; and recently, as a major tissue damaging process involved in the host response to severe acute respiratory syndrome coronavirus 2 infection. NETs are pertinent as a defence mechanism at the gut mucosal interphase exposed to high levels of bacteria, viruses and fungi. On the other hand, NETs can also potentiate and perpetuate gut inflammation. In this review, we discuss the broad protective vs. pathogenic roles of NETs, explanatory factors that could lead to an increase in NET formation in IBD and how NETs may contribute to gut inflammation and IBD-related complications. Finally, we summarise therapeutic opportunities to target NETs in IBD.
Assuntos
Armadilhas Extracelulares/imunologia , Doenças Inflamatórias Intestinais/imunologia , Neutrófilos/imunologia , Pesquisa Translacional Biomédica , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , COVID-19/imunologia , COVID-19/patologia , COVID-19/terapia , Humanos , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Neutrófilos/patologia , SARS-CoV-2RESUMO
Cystic fibrosis (CF) is a life-shortening, multi-organ, autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most prominent clinical manifestation in CF is the development of progressive lung disease characterised by an intense, chronic inflammatory airway response that culminates in respiratory failure and, ultimately, death. In recent years, a new class of therapeutics that have the potential to correct the underlying defect in CF, known as CFTR modulators, have revolutionised the field. Despite the exciting success of these drugs, their impact on airway inflammation, and its long-term consequences, remains undetermined. In addition, studies querying the absolute requirement for infection as a driver of CF inflammation have challenged the traditional consensus on CF pathogenesis, and also emphasise the need to prioritise complementary anti-inflammatory treatments in CF. Macrophages, often overlooked in CF research despite their integral role in other chronic inflammatory pathologies, have increasingly become recognised as key players in the initiation, perpetuation and resolution of CF lung inflammation, perhaps as a direct result of CFTR dysfunction. These findings suggest that macrophages may be an important target for novel anti-inflammatory interventional strategies to effectively treat CF lung function decline. This review will consider evidence for the efficacy of anti-inflammatory drugs in the treatment of CF, the potential role of macrophages, and the significance of targeting these pathways at a time when rectifying the basic defect in CF, through use of novel CFTR modulator therapies, is becoming increasingly viable.
Assuntos
Fibrose Cística , Anti-Inflamatórios/uso terapêutico , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Inflamação/tratamento farmacológico , MacrófagosRESUMO
The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (-10.1 ± 0.3 kcal mol-1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.
Assuntos
Quadruplex G , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Complexo Shelterina , Proteínas de Ligação a Telômeros/química , TermodinâmicaRESUMO
The structure of the 68 nt sequence with G-quadruplex forming potential within the hTERT promoter is disputed. One model features a structure with three stacked parallel G-quadruplex units, while another features an unusual duplex hairpin structure adjoined to two stacked parallel and antiparallel quadruplexes. We report here the results of an integrated structural biology study designed to distinguish between these possibilities. As part of our study, we designed a sequence with an optimized hairpin structure and show that its biophysical and biochemical properties are inconsistent with the structure formed by the hTERT wild-type sequence. By using circular dichroism, thermal denaturation, nuclear magnetic resonance spectroscopy, analytical ultracentrifugation, small-angle X-ray scattering, molecular dynamics simulations and a DNase I cleavage assay we found that the wild type hTERT core promoter folds into a stacked, three-parallel G-quadruplex structure. The hairpin structure is inconsistent with all of our experimental data obtained with the wild-type sequence. All-atom models for both structures were constructed using molecular dynamics simulations. These models accurately predicted the experimental hydrodynamic properties measured for each structure. We found with certainty that the wild-type hTERT promoter sequence does not form a hairpin structure in solution, but rather folds into a compact stacked three-G-quadruplex conformation.
Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Telomerase/genética , Sequência de Bases , Dicroísmo Circular , DNA/química , Humanos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The protein ALL1 fused from chromosome 1q (AF1q) is overexpressed in a variety of cancers and acts to activate several signaling pathways that lead to oncogenesis. For example, AF1q has been shown to interact with T-cell Factor 7 (TCF7; also known as TCF1) from the Wnt/ß-catenin pathway resulting in the transcriptional activation of the CD44 and the enhancement of breast cancer metastasis. Despite the importance of AF1q in facilitating oncogenesis and metastasis, the structural and biophysical properties of AF1q remain largely unexplored due to the absence of a viable method for producing recombinant protein. Here, we report the overexpression of AF1q in E. coli as a fusion to a N-terminal His6-tag, which forms inclusion bodies (IBs) during expression. The AF1q protein was purified from IBs under denaturing conditions by immobilized metal affinity chromatography followed by a successful one-step dialysis refolding. Refolded AF1q was further purified to homogeneity by gel filtration chromatography resulting in an overall yield of 35â¯mg/L culture. Our nuclear magnetic resonance (NMR) and analytical ultracentrifugation (AUC) measurements reveal AF1q interacts with TCF7, specifically with TCF7's high-mobility group (HMG) domain (residues 154-237), which is, to our knowledge, the first biophysical characterization of the AF1q and TCF7 interaction.
Assuntos
Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cromatografia de Afinidade , Clonagem Molecular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , UltracentrifugaçãoRESUMO
BACKGROUND: Defective macrophage phagolysosomal acidification is implicated in numerous lung diseases including Cystic Fibrosis (CF) and may contribute to defective pathogen killing. Conflicting reports relating to phagolysosomal pH in CF macrophages have been published, in part related to the use of pH-sensitive fluorescent probes where potential inadequacies in experimental design can be a contributing factor (e.g. employing probes with incorrect pKa for the cellular compartment of interest). We developed a reliable method to quantify macrophage phagolysosomal pH using surface-enhanced Raman spectroscopy-based nanosensors. METHODS: Monocyte-derived macrophages from CF and healthy control participants were incubated with nanosensors. Live cell imaging identified phagocytosed nanosensors, and surface-enhanced Raman spectroscopy was performed using para-mercaptobenzoic acid functionalised gold nanoparticles which produce Raman spectra that change predictably with their environmental pH. Conventional fluorescence spectroscopy was carried out in comparison. Nanosensor localisation to phagolysosomes was confirmed by transmission electron microscopy. RESULTS: Nanosensors were actively phagocytosed by macrophages into phagolysosomes and acidification occurred rapidly and remained stable for at least 60â¯min. There was no difference in phagolysosomal pH between healthy control and CF macrophages (5.41⯱â¯0.11 vs. 5.41⯱â¯0.20, pâ¯>â¯.9999), further confirmed by inhibiting Cystic Fibrosis Transmembrane Conductance Regulator in healthy control monocyte-derived macrophages. CONCLUSIONS: Optical nanosensors accurately measure macrophage phagolysosomal pH and demonstrate no phagolysosomal acidification defect in human CF monocyte-derived macrophages. Further studies using alveolar macrophages could extend the impact of our findings. Nanosensors represent a novel and precise means to measure organelle functions with widespread potential for the study and monitoring of several lung diseases.
Assuntos
Fibrose Cística , Macrófagos Alveolares , Fagossomos , Análise Espectral Raman , Adulto , Fenômenos Bioquímicos , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Macrófagos Alveolares/química , Macrófagos Alveolares/fisiologia , Masculino , Nanopartículas Metálicas , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Fagocitose , Fagossomos/química , Fagossomos/microbiologia , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodosRESUMO
Mammalian and bird ribosomes are nearly twice the mass of prokaryotic ribosomes in part because of their extraordinarily long rRNA tentacles. Human rRNA tentacles are not fully observable in current three-dimensional structures and their conformations remain to be fully resolved. In previous work we identified sequences that favor G-quadruplexes in silico and in vitro in rRNA tentacles of the human large ribosomal subunit. We demonstrated by experiment that these sequences form G-quadruplexes in vitro. Here, using a more recent motif definition, we report additional G-quadruplex sequences on surfaces of both subunits of the human ribosome. The revised sequence definition reveals expansive arrays of potential G-quadruplex sequences on LSU tentacles. In addition, we demonstrate by a variety of experimental methods that fragments of the small subunit rRNA form G-quadruplexes in vitro. Prior to this report rRNA sequences that form G-quadruplexes were confined to the large ribosomal subunit. Our combined results indicate that the surface of the assembled human ribosome contains numerous sequences capable of forming G-quadruplexes on both ribosomal subunits. The data suggest conversion between duplexes and G-quadruplexes in response to association with proteins, ions, or other RNAs. In some systems it seems likely that the integrated population of RNA G-quadruplexes may be dominated by rRNA, which is the most abundant cellular RNA.
Assuntos
Quadruplex G , RNA Mensageiro/química , RNA Ribossômico/química , Subunidades Ribossômicas/química , Ribossomos/química , Animais , Sequência de Bases , Humanos , Conformação de Ácido Nucleico , Filogenia , Homologia de SequênciaRESUMO
Analytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability, and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be used to characterize G4 formation and to monitor small-molecule binding.
Assuntos
Quadruplex G , Ultracentrifugação/métodos , Peso Molecular , Relação Estrutura-AtividadeRESUMO
To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.