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The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.
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Proteínas Recombinantes , Vibrio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Vibrio/genética , Vibrio/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , HumanosRESUMO
Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.
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Proteoma , Humanos , Acetilcoenzima A/química , LigantesRESUMO
Extensive inflammation causes epithelial cell hyperplasia in the airways and Bcl-2-interacting killer (Bik) reduces epithelial cell and mucous cell hyperplasia without affecting resting cells to restore homeostasis. These observations suggest that Bik induces apoptosis in a cell cycle-specific manner, but the mechanisms are not understood. Mice were exposed to an allergen for 3, 14, or 30 days and Bik expression was induced in airway epithelia of transgenic mice. Bik reduced epithelial and mucous cell hyperplasia when mice were exposed to an allergen for 3 or 14 days, but not when exposure lasted for 30 days, and Ki67-positivity was reduced. In culture, Bik expression killed proliferating cells but not quiescent cells. To capture the stage of the cell cycle when Bik induces cell death, airway cells that express fluorescent ubiquitin cell cycle indicators were generated that fluoresce red or green during the G0/G1 and S/G2/M phases of the cells cycle, respectively. Regardless of the cell cycle stage, Bik expression eliminated green-fluorescent cells. Also, Bik, when tagged with a blue-fluorescent protein, was only detected in green cells. Bik phosphorylation mutants at threonine 33 or serine 35 demonstrated that phosphorylation activated Bik to induce death even in quiescent cells. Immunoprecipitation and proteomic approaches identified casein kinase IIα to be responsible for phosphorylating and activating Bik to kill cells in S/G2/M. As casein kinase 2 alpha (CKIIα) is expressed only during the G2/M phase, we conclude that Bik activation in airway epithelial cells selectively targets hyperplastic epithelial cells, while leaving resting airway cells unaffected.
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Caseína Quinase II , Proteômica , Alérgenos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Divisão Celular , Hiperplasia , Camundongos , Proteínas Mitocondriais/metabolismoRESUMO
Background: Obesity is a global public health concern. Given the widespread disruption caused by the SARS-CoV-2 pandemic, it is important to evaluate its impact on children with chronic health conditions. This study examines the health of paediatric patients with obesity enrolled in a tertiary hospital weight management program, before and 1 year into the COVID-19 pandemic. Methods: This is a retrospective chart review of patients aged 2 to 17 years enrolled in a paediatric weight management clinic. Mental health outcomes (i.e., new referrals to psychologist, social work, eating disorder program, incidence of dysregulated eating, suicidal ideation, and/or self-harm) and physical health (anthropometric measures) were compared before and 1 year into the pandemic. Results: Among the 334 children seen in either period, there was an increase in referrals to psychologist (12.4% versus 26.5%; P=0.002) and the composite mental health outcome (17.2% versus 30.2%; P=0.005) during the pandemic compared with pre-pandemic. In a subset of children (n=30) with anthropometric measures in both periods, there was a lower rate of decline in BMIz score (-1.5 [2.00] versus -0.3 [0.73]/year; P=0.002) and an increase in adiposity (-0.8 [4.64] versus 2.7 [5.54]%/year; P=0.043) during the pandemic. Discussion: The pandemic has impacted the mental and physical health of children with obesity engaged in a weight management clinic. While our study provides evidence of a negative impact on mental health outcomes and less improvement in anthropometric measures, future research when patients return to in-person care will enable further examination of our findings with additional objective measures.
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OBJECTIVE: House dust mite (HDM) exposure is used to model experimental asthma in mice. However, a direct comparison of inflammatory responses following continuous versus intermittent HDM exposure has not been reported. Therefore, we investigated whether the HDM dose at sensitization or challenge affects extent of inflammation in mice that were either continuously or intermittently sensitized with HDM. MATERIALS AND METHODS: C57BL/6 mice received either 10 continuous exposures with 10 µg HDM per exposure or two intermittent HDM exposures over a period of two weeks and were subsequently challenged by three instillations with HDM during the third week. For the intermittent model, mice were sensitized with 1 or 10 µg HDM and challenged on three consecutive days with 1 or 10 µg HDM. Inflammatory cells in the bronchoalveolar lavage fluid and epithelial cell hyperplasia and mucous cell metaplasia were quantified. RESULTS: Significantly higher levels of inflammation and mucous cell metaplasia were observed when mice were sensitized intermittently compared with continuously. Intermittent sensitization and challenge with 10 µg HDM caused maximum inflammation, mucous cell metaplasia, and epithelial cell hyperplasia. However, sensitization with 1 µg HDM only also showed increased inflammation when challenged with 10 µg HDM. DISCUSSION: These findings suggest major differences in adaptive immunity, depending on the sensitization protocol. CONCLUSIONS: Because of significant differences, the HDM sensitization protocol should be carefully considered when designing studies to investigate the underlying mechanisms of immunity in mouse models of asthma.
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Alérgenos/administração & dosagem , Hipersensibilidade/imunologia , Inflamação/imunologia , Pyroglyphidae/imunologia , Imunidade Adaptativa , Alérgenos/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Células Epiteliais/patologia , Hiperplasia/patologia , Hipersensibilidade/patologia , Inflamação/patologia , Pulmão/imunologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.
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Glutarredoxinas/metabolismo , Hipersensibilidade/patologia , Hipersensibilidade/parasitologia , Pulmão/patologia , Pulmão/parasitologia , Pyroglyphidae/fisiologia , Animais , Citocinas/genética , Citocinas/metabolismo , Glutationa/metabolismo , Hiperplasia , Hipersensibilidade/sangue , Hipersensibilidade/complicações , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Muco/metabolismo , Pneumonia/sangue , Pneumonia/complicações , Pneumonia/parasitologia , Pneumonia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Hipersensibilidade Respiratória/sangue , Hipersensibilidade Respiratória/parasitologia , Hipersensibilidade Respiratória/patologia , Hipersensibilidade Respiratória/fisiopatologia , Mecânica Respiratória , Células Th2/imunologiaRESUMO
Mass gatherings are regarded as potential risks for transmission of infectious diseases, and might compromise the health system of countries in which they are hosted. The evidence for increased transmission of infectious diseases at international sporting mass gatherings that attract many visitors from all over the world is not clear, and the evidence base for public health surveillance, epidemiology, and response at events such as the Olympics is small. However, infectious diseases are a recognised risk, and public health planning is, and should remain, a crucial part of the overall planning of sporting events. In this Series paper, we set out the planning and the surveillance systems that were used to monitor public health risks during the London 2012 Olympic and Paralympic Games in the summer of 2012, and draw attention to the public health issues-infectious diseases and chemical, radiation, and environmental hazards-that arose. Although the absolute risk of health-protection problems, including infectious diseases, at sporting mass gatherings is small, the need for reassurance of the absence of problems is higher than has previously been considered; this could challenge conventional public health surveillance systems. Recognition of the limitations of health-surveillance systems needs to be part of the planning for future sporting events.
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Controle de Doenças Transmissíveis/organização & administração , Doenças Transmissíveis/epidemiologia , Vigilância em Saúde Pública/métodos , Esportes , Doenças Transmissíveis/transmissão , Aglomeração , Planejamento em Saúde/organização & administração , Humanos , Londres/epidemiologia , Síndrome , ViagemRESUMO
BACKGROUND: The National Human Papillomavirus (HPV) Vaccination Program in Australia commenced in 2007 for females and in 2013 for males, using the quadrivalent HPV vaccine (HPV 6,11,16,18). Thus far, we have demonstrated very substantial reductions in genital warts and in the prevalence of HPV among young Australian women, providing early evidence for the success of this public health initiative. Australia has a long history of school-based vaccination programs for adolescents, with comparatively high coverage. However, it is not clear what factors promote success in a school vaccination program. The HPV.edu study aims to examine: 1) student knowledge about HPV vaccination; 2) psycho-social outcomes and 3) vaccination uptake. METHODS/DESIGN: HPV.edu is a cluster randomised trial of a complex intervention in schools aiming to recruit 40 schools with year-8 enrolments above 100 students (approximately 4400 students). The schools will be stratified by Government, Catholic, and Independent sectors and geographical location, with up to 20 schools recruited in each of two states, Western Australia (WA) and South Australia (SA), and randomly allocated to intervention or control (usual practice). Intervention schools will receive the complex intervention which includes an adolescent intervention (education and distraction); a decisional support tool for parents and adolescents and logistical strategies (consent form returns strategies, in-school mop-up vaccination and vaccination-day guidelines). Careful process evaluation including an embedded qualitative evaluation will be undertaken to explore in depth possible mechanisms for any observed effect of the intervention on primary and secondary outcomes. DISCUSSION: This study is the first to evaluate the relative effectiveness of various strategies to promote best practice in school-based vaccination against HPV. The study aims to improve vaccination-related psychosocial outcomes, including adolescent knowledge and attitudes, decision-making involvement, self-efficacy, and to reduce fear and anxiety. The study also aims to improve school vaccination program logistics including reduction in time spent vaccinating adolescents and increased number of consent forms returned (regardless of decision). Less anxiety in adolescents will likely promote more efficient vaccination, which will be more acceptable to teachers, nurses and parents. Through these interventions, it is hoped that vaccination uptake will be increased. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry, ACTRN12614000404628 , 14.04.2014.
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Programas de Imunização , Papillomaviridae , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus , Aceitação pelo Paciente de Cuidados de Saúde , Instituições Acadêmicas , Vacinação , Adolescente , Adulto , Austrália , Tomada de Decisões , Emoções , Feminino , Educação em Saúde , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Infecções por Papillomavirus/virologia , Pais , Avaliação de Programas e Projetos de Saúde , Projetos de Pesquisa , Autoeficácia , Estudantes/psicologia , Adulto JovemRESUMO
The 2005 International Health Regulations (IHR) came into force for all Member States of the World Health Organization (WHO) in June 2007 and the deadline for achieving compliance was June 2012. The purpose of the IHR is to prevent, protect against, control - and provide a public health response to - international spread of disease. The territory of the United Kingdom of Great Britain and Northern Ireland and that of several other Member States, such as China, Denmark, France, the Netherlands and the United States of America, include overseas territories, which cover a total population of approximately 15 million people. Member States have a responsibility to ensure that all parts of their territory comply with the IHR. Since WHO has not provided specific guidance on compliance in the special circumstances of the overseas territories of Member States, compliance by these territories is an issue for self-assessment by Member States themselves. To date, no reports have been published on the assessment of IHR compliance in countries with overseas territories. We describe a gap analysis done in the United Kingdom to assess IHR compliance of its overseas territories. The findings and conclusions are broadly applicable to other countries with overseas territories which may have yet to assess their compliance with the IHR. Such assessments are needed to ensure compliance across all parts of a Member States' territory and to increase global health security.
Le Règlement sanitaire international de 2005 (RSI) est entré en vigueur pour tous les États membres de l'Organisation mondiale de la Santé en juin 2007, et la date limite pour sa mise en conformité était juin 2012. L'objectif du RSI est de prévenir, de protéger, de contrôler et d'apporter une réponse de santé publique à la propagation internationale des maladies. Le territoire du Royaume-Uni de Grande-Bretagne et d'Irlande du Nord et celui d'autres États membres, comme la Chine, le Danemark, la France, les Pays-Bas et les États-Unis d'Amérique, se composent de territoires d'outre-mer, lesquels couvrent une population totale d'environ 15 millions d'habitants. Les États membres ont la responsabilité de veiller à ce que toutes les parties de leur territoire se conforment au RSI. Étant donné que l'OMS ne fournit pas d'orientation spécifique concernant la conformité dans les circonstances spéciales des territoires d'outre-mer des États membres, leur conformité est une question d'auto-évaluation par les États membres eux-mêmes. À ce jour, aucun rapport n'a été publié sur l'évaluation de la conformité au RSI dans les pays possédant des territoires d'outre-mer. Nous décrivons une analyse des lacunes effectuée au Royaume-Uni pour évaluer la conformité au RSI de ses territoires d'outre-mer. Les résultats et les conclusions sont largement applicables aux autres pays possédant des territoires d'outre-mer, qui peuvent cependant évaluer leur propre conformité au RSI. Ces évaluations sont nécessaires pour veiller à la conformité dans toutes les parties du territoire d'un État membre et pour augmenter la sécurité sanitaire mondiale.
El Reglamento Sanitario Internacional 2005 (RSI) entró en vigor para todos los Estados miembros de la Organización Mundial de la Salud (OMS) en junio de 2007 con junio de 2012 como fecha límite para lograr el cumplimiento. El objetivo del RSI es prevenir, proteger, controlar y proporcionar una respuesta de salud pública a la propagación internacional de enfermedades. El territorio del Reino Unido de Gran Bretaña e Irlanda del Norte y otros Estados miembros como China, Dinamarca, Francia, los Países Bajos y los Estados Unidos de América cuentan con territorios de ultramar que abarcan una población total de aproximadamente 15 millones de personas. Los Estados miembros tienen la responsabilidad de garantizar que todos sus territorios cumplan con el RSI. Puesto que la OMS no ha proporcionado orientación específica sobre el cumplimiento para las circunstancias especiales de los territorios de ultramar de los Estados miembros, el cumplimiento por parte de estos territorios es un problema que los propios Estados miembros tienen que evaluar. Hasta la fecha no se han publicado informes sobre la evaluación del cumplimiento del RSI en los países con territorios de ultramar. Describimos un análisis de las deficiencias realizado en el Reino Unido con objeto de evaluar el cumplimiento del RSI de sus territorios de ultramar. Los resultados y conclusiones son ampliamente aplicables a otros países con territorios de ultramar que quizá aún tengan que evaluar su cumplimiento con el RSI. Dichas evaluaciones son necesarias para asegurar el cumplimiento en todos los territorios de los Estados miembros y para aumentar la seguridad sanitaria mundial.
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Controle de Doenças Transmissíveis/legislação & jurisprudência , Surtos de Doenças/prevenção & controle , Fidelidade a Diretrizes , Cooperação Internacional/legislação & jurisprudência , Implementação de Plano de Saúde , Promoção da Saúde/legislação & jurisprudência , Humanos , Irlanda do Norte/epidemiologia , Serviços Preventivos de Saúde/legislação & jurisprudência , Saúde Pública , Controle Social Formal , Reino Unido/epidemiologia , Organização Mundial da SaúdeRESUMO
Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1ß-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.
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Fibrose Cística , Criança , Lactente , Humanos , Camundongos , Animais , Fibrose Cística/complicações , Regulador de Condutância Transmembrana em Fibrose Cística , Propionatos , Bacteroides/genética , Células CACO-2 , Inflamação/complicações , Modelos Animais de Doenças , Disbiose/complicações , Escherichia coliRESUMO
Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within the cell or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein tissue inhibitor of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.
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Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.
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Antifúngicos , Aspergillus fumigatus , Farmacorresistência Fúngica , Ácidos Graxos Insaturados , Testes de Sensibilidade Microbiana , Triazóis , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Antifúngicos/farmacologia , Triazóis/farmacologia , Camundongos , Animais , Ácidos Graxos Insaturados/farmacologia , Humanos , Ensaios de Triagem em Larga Escala , Sinergismo Farmacológico , Rhizopus/efeitos dos fármacos , Rhizopus/genética , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Piperidinas/farmacologia , Modelos Animais de Doenças , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Aspergilose Pulmonar Invasiva/microbiologiaRESUMO
Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.
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Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Linhagem Celular Tumoral , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases/metabolismoRESUMO
OBJECTIVE: Cardiovascular disease (CVD) is the leading disease burden in Aboriginal Australians, but culturally appropriate cardiac rehabilitation programs are lacking. We evaluated the uptake and effects on lifestyle, and cardiovascular risk factors, of cardiac rehabilitation at an Aboriginal Medical Service (AMS). METHODS: The program involved weekly exercise and education sessions (through 'yarning') for Aboriginal people with or at risk of CVD. Participants' perceptions of the program and the impact on risk factors were evaluated following 8 weeks of attendance. RESULTS: In twenty-eight participants (20 females) who completed 8 weeks of sessions, body mass index (34.0 ± 5.1 v. 33.3 ± 5.2 kgm⻲; P<0.05), waist girth (113 ± 14 v. 109 ± 13 cm; P<0.01) and blood pressure (135/78 ± 20/12 v. 120/72 ± 16/5 mmHg; P<0.05) decreased and 6- min walk distance increased (296 ± 115 v. 345 ± 135m; P<0.01). 'Yarning' helped identify and address a range of chronic health issues including medication compliance, risk factor review and chest pain management. CONCLUSIONS: AMS-based cardiac rehabilitation was well attended, and improved cardiovascular risk factors and health management. An AMS is an ideal location for managing cardiovascular health and provides a setting conducive to addressing a broad range of chronic conditions.
Assuntos
Institutos de Cardiologia/estatística & dados numéricos , Reabilitação Cardíaca , Exercício Físico/fisiologia , Educação em Saúde/métodos , Serviços de Saúde do Indígena/estatística & dados numéricos , Havaiano Nativo ou Outro Ilhéu do Pacífico/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Austrália , Índice de Massa Corporal , Doenças Cardiovasculares/etnologia , Doenças Cardiovasculares/prevenção & controle , Competência Cultural , Feminino , Grupos Focais , Serviços de Saúde do Indígena/tendências , Humanos , Hipertensão/complicações , Hipertensão/etnologia , Hipertensão/terapia , Masculino , Pessoa de Meia-Idade , Desenvolvimento de Programas/métodos , Avaliação de Programas e Projetos de Saúde , Fatores de Risco , Adulto JovemRESUMO
IMPORTANCE: PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.
Assuntos
Fibrose Cística , Micoses , Humanos , Aspergillus fumigatus , Fibrose Cística/microbiologia , Parede Celular , BiofilmesRESUMO
Although chronic low-grade inflammation does not cause immediate clinical symptoms, over the longer term, it can enhance other insults or age-dependent damage to organ systems and thereby contribute to age-related disorders, such as respiratory disorders, heart disease, metabolic disorders, autoimmunity, and cancer. However, the molecular mechanisms governing low-level inflammation are largely unknown. We discovered that Bcl-2-interacting killer (Bik) deficiency causes low-level inflammation even at baseline and the development of spontaneous emphysema in female but not male mice. Similarly, a single nucleotide polymorphism that reduced Bik levels was associated with increased inflammation and enhanced decline in lung function in humans. Transgenic expression of Bik in the airways of Bik-deficient mice inhibited allergen- or LPS-induced lung inflammation and reversed emphysema in female mice. Bik deficiency increased nuclear but not cytosolic p65 levels because Bik, by modifying the BH4 domain of Bcl-2, interacted with regulatory particle non-ATPase 1 (RPN1) and RPN2 and enhanced proteasomal degradation of nuclear proteins. Bik deficiency increased inflammation primarily in females because Bcl-2 and Bik levels were reduced in lung tissues and airway cells of female compared with male mice. Therefore, controlling low-grade inflammation by modifying the unappreciated role of Bik and Bcl-2 in facilitating proteasomal degradation of nuclear proteins may be crucial in treating chronic age-related diseases.
Assuntos
Enfisema , Hexosiltransferases , Masculino , Animais , Feminino , Humanos , Camundongos , Apoptose , Proteínas Mitocondriais , Proteínas Reguladoras de Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Inflamação/genética , Proteínas Nucleares , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
Aspergillus fumigatus is a human fungal pathogen that is most often avirulent in immunecompetent individuals because the innate immune system is efficient at eliminating fungal conidia. However, recent clinical observations have shown that severe influenza A virus (IAV) infection can lead to secondary A. fumigatus infections with high mortality. Little is currently known about how IAV infection alters the innate antifungal immune response. Here, we established a murine model of IAV-induced A. fumigatus (IAV-Af) superinfection by inoculating mice with IAV followed 6 days later by A. fumigatus conidia challenge. We observed increased mortality in the IAV-Af-superinfected mice compared to mice challenged with either IAV or A. fumigatus alone. A. fumigatus conidia were able to germinate and establish a biofilm in the lungs of the IAV-Af superinfection group, which was not seen following fungal challenge alone. While we did not observe any differences in inflammatory cell recruitment in the IAV-Af superinfection group compared to single-infection controls, we observed defects in Aspergillus conidial uptake and killing by both neutrophils and monocytes after IAV infection. pHrodo Green zymosan bioparticle (pHrodo-zymosan) and CM-H2DCFDA [5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate] staining, indicators of phagolysosome maturation and reactive oxygen species (ROS) production, respectively, revealed that the fungal killing defect was due in part to reduced phagolysosome maturation. Collectively, our data demonstrate that the ability of neutrophils and monocytes to kill and clear Aspergillus conidia is strongly reduced in the pulmonary environment of an IAV-infected lung, which leads to invasive pulmonary aspergillosis and increased overall mortality in our mouse model, recapitulating what is observed clinically in humans. IMPORTANCE Influenza A virus (IAV) is a common respiratory virus that causes seasonal illness in humans, but can cause pandemics and severe infection in certain patients. Since the emergence of the 2009 H1N1 pandemic strains, there has been an increase in clinical reports of IAV-infected patients in the intensive care unit (ICU) developing secondary pulmonary aspergillosis. These cases of flu-Aspergillus superinfections are associated with worse clinical outcomes than secondary bacterial infections in the setting of IAV. To date, we have a limited understanding of the cause(s) of secondary fungal infections in immunocompetent hosts. IAV-induced modulation of cytokine production and innate immune cellular function generates a unique immune environment in the lung, which could make the host vulnerable to a secondary fungal infection. Our work shows that defects in phagolysosome maturation in neutrophils and monocytes after IAV infection impair the ability of these cells to kill A. fumigatus, thus leading to increased fungal germination and growth and subsequent invasive aspergillosis. Our work lays a foundation for future mechanistic studies examining the exact immune modulatory events occurring in the respiratory tract after viral infection leading to secondary fungal infections.
Assuntos
Aspergilose , Vírus da Influenza A Subtipo H1N1 , Superinfecção , Humanos , Animais , Camundongos , Aspergillus fumigatus , Esporos Fúngicos , Zimosan , Aspergilose/microbiologia , AspergillusRESUMO
OBJECTIVE: Assess the impact of introducing a consensus guideline incorporating an adapted Sepsis Risk Calculator (SRC) algorithm, in the management of early onset neonatal sepsis (EONS), on antibiotic usage and patient safety. DESIGN: Multicentre prospective study SETTING: Ten perinatal hospitals in Wales, UK. PATIENTS: All live births ≥34 weeks' gestation over a 12-month period (April 2019-March 2020) compared with infants in the preceding 15-month period (January 2018-March 2019) as a baseline. METHODS: The consensus guideline was introduced in clinical practice on 1 April 2019. It incorporated a modified SRC algorithm, enhanced in-hospital surveillance, ongoing quality assurance, standardised staff training and parent education. The main outcome measure was antibiotic usage/1000 live births, balancing this with analysis of harm from delayed diagnosis and treatment, disease severity and readmissions from true sepsis. Outcome measures were analysed using statistical process control charts. MAIN OUTCOME MEASURES: Proportion of antibiotic use in infants ≥34 weeks' gestation. RESULTS: 4304 (14.3%) of the 30 105 live-born infants received antibiotics in the baseline period compared with 1917 (7.7%) of 24 749 infants in the intervention period (45.5% mean reduction). All 19 infants with culture-positive sepsis in the postimplementation phase were identified and treated appropriately. There were no increases in sepsis-related neonatal unit admissions, disease morbidity and late readmissions. CONCLUSIONS: This multicentre study provides evidence that a judicious adaptation of the SRC incorporating enhanced surveillance can be safely introduced in the National Health Service and is effective in reducing antibiotic use for EONS without increasing morbidity and mortality.
Assuntos
Sepse Neonatal , Sepse , Algoritmos , Antibacterianos/uso terapêutico , Feminino , Humanos , Lactente , Recém-Nascido , Sepse Neonatal/diagnóstico , Sepse Neonatal/tratamento farmacológico , Sepse Neonatal/epidemiologia , Gravidez , Estudos Prospectivos , Medição de Risco , Sepse/diagnóstico , Sepse/tratamento farmacológico , Sepse/epidemiologia , Medicina Estatal , País de GalesRESUMO
BACKGROUND: Tuberculosis (TB) in migrants is an ongoing challenge in several low TB incidence countries since a large proportion of TB in these countries occurs in migrants from high incidence countries. To meet these challenges, several countries utilize TB screening programs. The programs attempt to identify and treat those with active and/or infectious stages of the disease. In addition, screening is used to identify and manage those with latent or inactive disease after arrival. Between nations, considerable variation exists in the methods used in migration-associated TB screening. The present study aimed to compare the TB immigration medical examination requirements in selected countries of high immigration and low TB incidence rates. METHODS: Descriptive study of immigration TB screening programs. RESULTS: 16 out of 18 eligible countries responded to the written standardized survey and phone interview. Comparisons in specific areas of TB immigration screening programs included authorities responsible for TB screening, the primary objectives of the TB screening program, the yield of detection of active TB disease, screening details and aspects of follow up for inactive pulmonary TB. No two countries had the same approach to TB screening among migrants. Important differences, common practices, common problems, evidence or lack of evidence for program specifics were noted. CONCLUSIONS: In spite of common goals, there is great diversity in the processes and practices designed to mitigate the impact of migration-associated TB among nations that screen migrants for the disease. The long-term goal in decreasing migration-related introduction of TB from high to low incidence countries remains diminishing the prevalence of the disease in those high incidence locations. In the meantime, existing or planned migration screening programs for TB can be made more efficient and evidenced based. Cooperation among countries doing research in the areas outlined in this study should facilitate the development of improved screening programs.