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2.
J Vis Exp ; (205)2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38587373

RESUMO

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.


Assuntos
AMP Cíclico , Sistema Respiratório , Humanos , AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Colforsina/farmacologia , Células HEK293 , Sistema Respiratório/metabolismo
3.
PLoS One ; 19(4): e0300285, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38564604

RESUMO

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®â€¢GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®â€¢GUT will yield more representative microbial communities.


Assuntos
DNA , Manejo de Espécimes , Bovinos , Humanos , Animais , Manejo de Espécimes/métodos , Fezes/química , DNA/análise , Etanol/análise , Sistema Respiratório , Genômica , RNA Ribossômico 16S/genética
5.
Angle Orthod ; 94(3): 328-335, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38639454

RESUMO

OBJECTIVES: To evaluate maximal inspiratory (MIP) and expiratory (MEP) pressures, which are reflective of respiratory muscle strength, in skeletal Class II patients with different growth patterns (horizontal, average, and vertical) and to correlate those with airway dimension. MATERIALS AND METHODS: Patients with a Class II skeletal base seeking orthodontic treatment were assigned to the following groups: average, horizontal, and vertical growth pattern. The control group (n = 14) comprised patients with a Class I skeletal base and average growth pattern. Airway dimensions were obtained using cone-beam computed tomography scans, and a spirometer with a pressure transducer was used for assessment of MIP and MEP. Routine spirometry for assessment of lung function was also performed. RESULTS: No significant differences were found in maximal inspiratory and expiratory pressures for the study groups in comparison with the control group. Class I patients had significantly greater oropharyngeal and nasopharyngeal airway volumes compared with the study groups. No significant difference in minimal cross-section area of the airway was observed among groups. A weak positive correlation between maximal inspiratory pressure and airway volume was observed. CONCLUSIONS: Although Class I patients displayed significantly greater oropharyngeal and nasopharyngeal airway volumes, there was no significant difference in respiratory muscle strength or airway function between Class II patients with different growth patterns and the Class I control group. The findings underscore the significance of exploring factors beyond craniofacial growth patterns that may contribute to sleep-related breathing disorders.


Assuntos
Nasofaringe , Sistema Respiratório , Humanos , Orofaringe/diagnóstico por imagem , Músculos Respiratórios , Respiração , Tomografia Computadorizada de Feixe Cônico/métodos
6.
Clin Oral Investig ; 28(5): 252, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38627272

RESUMO

OBJECTIVE: Craniofacial anomalies are widely discussed as predisposing factors of breathing disorders. Since many more cofactors exist, this study investigated the association between maxillary micrognathia and morphological changes of posterior airway space and adenoids in these patients. MATERIAL AND METHODS: Cephalometric radiographs of n = 73 patients were used for data acquisition. The patients were divided into two groups according to certain skeletal characteristics: maxillary micrognathia (n = 34, 16 female, 18 male; mean age 10.55 ± 3.03 years; defined by a SNA angle < 79°) and maxillary eugnathia (n = 39, 19 female, 20 male; mean age 10.93 ± 3.26 years; defined by a SNA angle > 79°). The evaluation included established procedures for measurements of the maxilla, posterior airway space and adenoids. Statistics included Kolmogorov-Smirnov-, T- and Mann-Whitney-U-Tests for the radiographs. The level of significance was set at p < 0.05. RESULTS: The cephalometric analysis showed differences in the superior posterior face height and the depth of the posterior airway space at palatal level among the two groups. The depth of the posterior airway space at mandibular level was the same for both groups, just as the size of the area taken by adenoids in the nasopharynx. CONCLUSIONS: Skeletal anomalies affect the dimension of the posterior airway space. There were differences among the subjects with maxillary micrognathia and these with a normal maxilla. However, the maxilla was only assessed in the sagittal direction, not in the transverse. This study showed that the morphology of the maxilla relates to the posterior airway space whereas the adenoids seem not to be affected. CLINICAL RELEVANCE: Maxillary micrognathia is significantly associated with a smaller depth of the posterior airway space at the palatal level compared to patients with maxillary eugnathia.


Assuntos
Tonsila Faríngea , Micrognatismo , Humanos , Masculino , Feminino , Criança , Adolescente , Micrognatismo/diagnóstico por imagem , Nasofaringe , Maxila/diagnóstico por imagem , Sistema Respiratório , Cefalometria/métodos
9.
Sci Rep ; 14(1): 9056, 2024 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-38643191

RESUMO

The impact of evolving treatment regimens, airway clearance strategies, and antibiotic combinations on the incidence and prevalence of respiratory infection in cystic fibrosis (CF) in children and adolescents remains unclear. The incidence, prevalence, and prescription trends from 2002 to 2019 with 18,339 airway samples were analysed. Staphylococcus aureus [- 3.86% (95% CI - 5.28-2.43)] showed the largest annual decline in incidence, followed by Haemophilus influenzae [- 3.46% (95% CI - 4.95-1.96)] and Pseudomonas aeruginosa [- 2.80%95% CI (- 4.26-1.34)]. Non-tuberculous mycobacteria and Burkholderia cepacia showed a non-significant increase in incidence. A similar pattern of change in prevalence was observed. No change in trend was observed in infants < 2 years of age. The mean age of the first isolation of S. aureus (p < 0.001), P. aeruginosa (p < 0.001), H. influenza (p < 0.001), Serratia marcescens (p = 0.006) and Aspergillus fumigatus (p = 0.02) have increased. Nebulised amikacin (+ 3.09 ± 2.24 prescription/year, p = 0.003) and colistin (+ 1.95 ± 0.3 prescriptions/year, p = 0.032) were increasingly prescribed, while tobramycin (- 8.46 ± 4.7 prescriptions/year, p < 0.001) showed a decrease in prescription. Dornase alfa and hypertonic saline nebulisation prescription increased by 16.74 ± 4.1 prescriptions/year and 24 ± 4.6 prescriptions/year (p < 0.001). There is a shift in CF among respiratory pathogens and prescriptions which reflects the evolution of cystic fibrosis treatment strategies over time.


Assuntos
Fibrose Cística , Pneumonia , Infecções por Pseudomonas , Criança , Lactente , Humanos , Adolescente , Fibrose Cística/complicações , Fibrose Cística/epidemiologia , Fibrose Cística/microbiologia , Staphylococcus aureus , Sistema Respiratório/microbiologia , Antibacterianos/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Pneumonia/tratamento farmacológico , Pseudomonas aeruginosa
10.
Science ; 384(6691): 30-31, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38574157

RESUMO

Bronchoconstriction causes epithelial cell extrusion that promotes airway inflammation.


Assuntos
Asma , Broncoconstrição , Humanos , Sistema Respiratório , Inflamação , Células Epiteliais
11.
Artigo em Inglês | MEDLINE | ID: mdl-38524398

RESUMO

Purpose: The heterogeneity of clinical features in COPD at stable state has been associated with airway microbiota. Blood eosinophil count (BEC) represents a biomarker for a pejorative evolution of COPD, including exacerbations and accelerated FEV1 decline. We aimed to analyse the associations between BEC and airway microbiota in COPD at stable state. Patients and Methods: Adult COPD patients at stable state (RINNOPARI cohort) were included and characterised for clinical, functional, biological and morphological features. BEC at inclusion defined 2 groups of patients with low BEC <300/mm3 and high BEC ≥300/mm3. Sputa were collected and an extended microbiological culture was performed for the identification of viable airway microbiota. Results: Fifty-nine subjects were included. When compared with the low BEC (n=40, 67.8%), the high BEC group (n=19, 32.2%) had more frequent exacerbations (p<0.001) and more pronounced cough and sputum (p<0.05). The global composition, the number of bacteria per sample and the α-diversity of the microbiota did not differ between groups, as well as the predominant phyla (Firmicutes), or the gender repartition. Conclusion: In our study, high BEC in COPD at stable state was associated with a clinical phenotype including frequent exacerbation, but no distinct profile of viable airway microbiota compared with low BEC.


Assuntos
Eosinofilia , Microbiota , Doença Pulmonar Obstrutiva Crônica , Adulto , Humanos , Eosinófilos , Progressão da Doença , Sistema Respiratório , Contagem de Leucócitos , Escarro/microbiologia
13.
Environ Toxicol ; 39(5): 3264-3273, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38459623

RESUMO

Bisphenol A (BPA) is a substance that can harm the environment and human health by interfering with the normal functioning of the body's hormonal system. It is commonly found in various plastic-based products such as cosmetics, canned foods, beverage containers, and medical equipment and as well as it can also be absorbed by inhalation. There have been limited studies on the effects of BPA on lung fibroblasts, and it is still unclear how high levels of BPA can impact respiratory system cells, particularly the lungs and trachea. In this research, we aimed to investigate the cell cycle disruption potential of BPA on respiratory system cells by examining healthy trachea and lung cells together for the first time. The findings indicated that BPA exposure can alter the healthy cells' morphology, leading to reduced cellular viability that has been assessed by MTT and SRB assays. BPA treatment was able to activate caspase3 as expected, which could cause apoptosis in treated cells. Although the highest dose of BPA did not increase the apoptotic rate of rat trachea cells, it remarkably caused them to become necrotic (52.12%). In addition to quantifying the induction of apoptosis and necrosis by BPA, cell cycle profiles were also determined using flow cytometry. Thereby, BPA treatment unexpectedly inhibited the cell cycle's progression by causing G2/M cell cycle arrest in both lung and tracheal cells, which hindered cell proliferation. The findings of the study suggested that exposure to BPA could lead to serious respiratory problems, even respiratory tract cancers via alterations in the cell cycle.


Assuntos
Apoptose , Compostos Benzidrílicos , Fenóis , Ratos , Animais , Humanos , Morte Celular , Proliferação de Células , Compostos Benzidrílicos/toxicidade , Pontos de Checagem da Fase G2 do Ciclo Celular , Sistema Respiratório
14.
Environ Sci Technol ; 58(14): 6083-6092, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38547129

RESUMO

Despite significant advances in understanding the general health impacts of air pollution, the toxic effects of air pollution on cells in the human respiratory tract are still elusive. A robust, biologically relevant in vitro model for recapitulating the physiological response of the human airway is needed to obtain a thorough understanding of the molecular mechanisms of air pollutants. In this study, by using 1-nitropyrene (1-NP) as a proof-of-concept, we demonstrate the effectiveness and reliability of evaluating environmental pollutants in physiologically active human airway organoids. Multimodal imaging tools, including live cell imaging, fluorescence microscopy, and MALDI-mass spectrometry imaging (MSI), were implemented to evaluate the cytotoxicity of 1-NP for airway organoids. In addition, lipidomic alterations upon 1-NP treatment were quantitatively analyzed by nontargeted lipidomics. 1-NP exposure was found to be associated with the overproduction of reactive oxygen species (ROS), and dysregulation of lipid pathways, including the SM-Cer conversion, as well as cardiolipin in our organoids. Compared with that of cell lines, a higher tolerance of 1-NP toxicity was observed in the human airway organoids, which might reflect a more physiologically relevant response in the native airway epithelium. Collectively, we have established a novel system for evaluating and investigating molecular mechanisms of environmental pollutants in the human airways via the combinatory use of human airway organoids, multimodal imaging analysis, and MS-based analyses.


Assuntos
Poluentes Atmosféricos , Pirenos , Sistema Respiratório , Humanos , Reprodutibilidade dos Testes , Organoides , Imagem Multimodal
16.
Euro Surveill ; 29(11)2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38487888

RESUMO

BackgroundSurveillance of lower respiratory tract infections (LRTI) of operated patients conventionally focuses on intubated patients in intensive care units (ICU). Post-operative immobilisation increases the risk of LRTI not associated with ventilators. Operated patients, however, have thus far not been a primary target for LRTI surveillance.AimWe aimed to describe the applied LRTI surveillance method in the German surveillance module for operated patients (OP-KISS) and to report data between 2018 and 2022.MethodsSurveillance of LRTI can be performed voluntarily in addition to surgical site infection (SSI) surveillance in OP-KISS. We calculated LRTI rates per 100 operations for all procedures combined, as well as for individual surgical groups and procedures. Additionally, a combined post-operative infection rate (SSI and LRTI) was calculated.ResultsSurveillance of LRTI was performed in 4% of all participating OP-KISS departments and for 2% (23,239 of 1,332,438) of all procedures in the OP-KISS database. The pooled LRTI rate was 0.9 per 100 operations, with marked differences between different types of surgery (3.6 for lobectomies, 0.1 for traumatology and orthopaedics). The share of LRTI among all post-operative infections was highly variable. For lobectomies, the LRTI rate was higher than the SSI rate (3.6 vs 1.5 per 100 operations).ConclusionSurveillance of post-operative LRTI is not yet widely adopted by German hospitals. Based on the data in this study, lobectomies represent a prime target for post-operative LRTI surveillance.


Assuntos
Infecção Hospitalar , Infecções Respiratórias , Humanos , Infecção Hospitalar/epidemiologia , Vigilância da População/métodos , Infecção da Ferida Cirúrgica/epidemiologia , Unidades de Terapia Intensiva , Infecções Respiratórias/epidemiologia , Sistema Respiratório
20.
Cells ; 13(6)2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38534319

RESUMO

Chronic Obstructive Pulmonary Disease (COPD) is a pathological condition of the respiratory system characterized by chronic airflow obstruction, associated with changes in the lung parenchyma (pulmonary emphysema), bronchi (chronic bronchitis) and bronchioles (small airways disease). In the last years, the importance of phenotyping and endotyping COPD patients has strongly emerged. Metabolomics refers to the study of metabolites (both intermediate or final products) and their biological processes in biomatrices. The application of metabolomics to respiratory diseases and, particularly, to COPD started more than one decade ago and since then the number of scientific publications on the topic has constantly grown. In respiratory diseases, metabolomic studies have focused on the detection of metabolites derived from biomatrices such as exhaled breath condensate, bronchoalveolar lavage, and also plasma, serum and urine. Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy are powerful tools in the precise identification of potentially prognostic and treatment response biomarkers. The aim of this article was to comprehensively review the relevant literature regarding the applications of metabolomics in COPD, clarifying the potential clinical utility of the metabolomic profile from several biologic matrices in detecting biomarkers of disease and prognosis for COPD. Meanwhile, a complete description of the technological instruments and techniques currently adopted in the metabolomics research will be described.


Assuntos
Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Sistema Respiratório/metabolismo , Metabolômica/métodos , Biomarcadores/metabolismo , Espectrometria de Massas/métodos
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