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BACKGROUND: The prevalence of fungi in cystic fibrosis (CF) lung infections is poorly understood and studies have focused on adult patients. We investigated the fungal diversity in children with CF using bronchoalveolar lavage (BAL) and induced sputum (IS) samples to capture multiple lung niches. METHODS: Sequencing of the fungal ITS2 region and molecular mycobiota diversity analysis was performed on 25 matched sets of BAL-IS samples from 23 children collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). RESULTS: Aspergillus and Candida were detected in all samples and were the most abundant and prevalent genera, followed by Dipodascus, Lecanicillium and Simplicillium. The presumptive CF pathogens Exophiala, Lomentospora and Scedosporium were identified at variable abundances in 100 %, 64 %, and 24 % of sample sets, respectively. Fungal pathogens observed at high relative abundance (≥40 %) were not accurately diagnosed by routine culture microbiology in over 50 % of the cohort. The fungal communities captured by BAL and IS samples were similar in diversity and composition, with exception to C. albicans being significantly increased in IS samples. The respiratory mycobiota varied greatly between individuals, with only 13 of 25 sample sets containing a dominant fungal taxon. In 11/25 BAL sample sets, airway compartmentalisation was observed with diverse mycobiota detected from different lobes of the lung. CONCLUSIONS: The paediatric mycobiota is diverse, complex and inadequately diagnosed by conventional microbiology. Overlapping fungal communities were identified in BAL and IS samples, showing that IS can capture fungal genera associated with the lower airway. Compartmentalisation of the lower airway presents difficulties for consistent mycobiota sampling.
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Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.
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Proteínas de Drosophila , Complejo Represivo Polycomb 1 , Animales , Sistema Nervioso Central/metabolismo , Cromatina/metabolismo , Cromatina/genética , Drosophila/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Larva/metabolismo , Larva/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/genética , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred â¼1â h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.
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Calcio , Timerosal , Ratones , Masculino , Animales , Timerosal/farmacología , Semen , Espermatozoides/fisiología , Adenosina Trifosfato , Fertilización/fisiologíaRESUMEN
The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiología , Aguas Residuales , Pandemias , Monitoreo Epidemiológico Basado en Aguas Residuales , Pruebas en el Punto de AtenciónRESUMEN
BACKGROUND: Spatial topography of the cystic fibrosis (CF) lung microbiota is poorly understood in childhood. How best to sample the respiratory tract in children for microbiota analysis, and the utility of microbiota profiling in clinical management of early infection remains unclear. By comparison with bronchoalveolar lavage (BAL), we assessed the ability of induced sputum (IS) sampling to characterise the lower airway microbiota. METHODS: Sample sets from IS and two or three matched BAL compartments were obtained for microbiota analysis as part of the CF-Sputum Induction Trial (UKCRN_14615, ISRCTNR_12473810). Microbiota profiles and pathogen detection were compared between matched samples. RESULTS: Twenty-eight patients, aged 1.1-17.7 years, provided 30 sample sets. Within-patient BAL comparisons revealed spatial heterogeneity in 8/30 (27%) sample sets indicating that the lower airway microbiota from BAL is frequently compartmentalised in children with CF. IS samples closely resembled one or more matched BAL compartments in 15/30 (50%) sets, and were related in composition in a further 9/30 (30%). IS detected 86.2% of the Top 5 genera found across matched BAL samples. The sensitivity of IS to detect specific CF-pathogens identified in matched BAL samples at relative abundance ≥5% varied between 43 and 100%, with negative predictive values between 73 and 100%. CONCLUSIONS: Spatial heterogeneity of the lower airway microbiota was observed in BAL samples and presents difficulties for consistent lung sampling. IS captured a microbiota signature representative of the lower airway in 80% of cases, and is a straightforward, non-invasive intervention that can be performed frequently to aid pathogen diagnosis and understand microbiota evolution in children with CF.