Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ.

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.

Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

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.

Assessment of maximal inspiratory and expiratory pressures in skeletal Class II patients with different growth patterns.

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.

Comparison of PAS and adenoids in patients with and without maxillary micrognathia before orthodontic treatment.

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.

The changing epidemiology of pulmonary infection in children and adolescents with cystic fibrosis: an 18-year experience.

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.

Epithelial cells crowded out in asthma.

Bronchoconstriction causes epithelial cell extrusion that promotes airway inflammation.

High Blood Eosinophil Count at Stable State is Not Associated with Airway Microbiota Distinct Profile in COPD.

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.

Bisphenol A promotes cell death in healthy respiratory system cells through inhibition of cell proliferation and induction of G2/M cell cycle arrest.

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.

Human Airway Organoids and Multimodal Imaging-Based Toxicity Evaluation of 1-Nitropyrene.

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.

Surveillance of infections of surgical sites and lower respiratory tracts should be combined: experiences from the German surveillance module for operated patients (OP-KISS), 2018 to 2022.

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.

Exploring the Potential Role of Metabolomics in COPD: A Concise Review.

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.

[Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems]. / Les co-infections virales affectant le système respiratoire porcin : conséquences et limites des systèmes d'étude.

Coinfections affecting the porcine respiratory system have often been overlooked, in favor of mono-infections, even though they are significantly more common in the field. In pigs, the term 'porcine respiratory complex' is used to describe coinfections involving both viruses, such as, for example, the swine influenza type A virus (swIAV), the porcine respiratory and reproductive syndrome virus (PRRSV), and the porcine circovirus type 2 (PCV-2), as well as bacteria. Until recently, most studies were primarily focused on clinical aspects and paid little attention to the molecular consequences of coinfections. This narrative review addresses the consequences of coinfections in the porcine respiratory system involving viruses. When possible, interactions that can occur between viruses are briefly presented. Conversely, research involving bacteria, protozoa, and fungi has not been considered at all. Finally, the main limitations complicating the interpretation of results from coinfection/superinfection studies are considered, and prospects in this exciting field of health research are presented.

A RETROSPECTIVE EVALUATION OF MORBIDITY AND MORTALITY IN ASSOCIATION WITH INFESTATION BY THE MITE STERNOSTOMA TRACHEACOLUM IN AN AVIARY-HOUSED POPULATION OF GOULDIAN FINCHES (CHLOEBIA GOULDIAE).

The Rhynonyssid mesostigmatic mite, Sternostoma tracheacolum, is a well-documented endoparasitic hematophagous arthropod of the respiratory tracts of multiple avian species, particularly Estrildid finches and canaries. In this retrospective study, 175 medical and 278 pathology records for the Gouldian finch (Chloebia gouldiae) population (N = 377) at the San Diego Zoo between 2013 and 2021 were analyzed to evaluate the effectiveness of ivermectin-based prophylaxis. A multivariable negative binomial regression model was constructed to evaluate the population effects of monthly treatments on morbidity or mortality associated with respiratory mites. While controlling for other factors in the model, the prophylactic treatment did not significantly reduce the monthly rate of mite-associated morbidity or mortality (IRR = 1.017, 95% CI: 0.997-1.036, P = 0.0759); however, low proportions of the population were prophylactically treated over time. Different factors were significant when separately evaluating adjusted associations with respiratory morbidity and mortality. The findings suggest increased rates of respiratory morbidity for each successive year of the study period (IRR = 1.180, 95% CI: 1.046-1.342, P = 0.0090) and increased rates of mite-associated mortality occurring annually between May and October (IRR = 1.697, 95% CI: 1.034-2.855, P = 0.0404) compared to the wet winter season. Our findings highlight the need to continually evaluate and optimize treatment regimens in zoological collections. Further investigations into this host-parasite relationship and potential treatments and preventive therapies are warranted.

SARS-CoV-2 infection increases airway bleeding risk in patients after tracheostomies.

BACKGROUND: Airway bleeding events are a rare incident in SARS-CoV-2-infected patients after tracheostomies. We aimed to explore the correlation between airway bleeding and SARS-CoV-2 infection and evaluate the consistency of SARS-CoV-2 RNA test results in the upper and lower airway samples from patients after tracheostomies. METHODS: Forty-four patients after temporary or permanent tracheostomy were divided into a positive group (29 patients) and a negative group (15 patients) based on the SARS-CoV-2 RNA test results of their oropharyngeal swabs. The oropharyngeal and tracheal swabs of the positive group were re-collected for SARS-CoV-2 RNA detection. Demographic and clinical characteristics and airway bleeding events were recorded for all enrolled patients. RESULTS: Airway bleeding was reported in eleven patients of the positive group (11/29), with seven displaying bloody sputum or hemoptysis, and four featuring massive sputum crust formation in the trachea that resulted in dyspnea, and only one patient in the negative group (1/15), with a significant difference in the airway bleeding rate (37.9% vs. 6.7%, p < 0.05). The SARS-CoV-2 RNA test results showed a statistical difference in cycle threshold (Ct) values between oropharyngeal swabs and tracheal swabs (p < 0.05). CONCLUSIONS: After tracheostomies, patients are more susceptible to airway bleeding if they are infected with SARS-CoV-2. The findings signify that in addition to droplet transmission through tracheostoma, SARS-CoV-2 may infect the oropharynx by airborne and close contact transmission, and that given the higher viral load and longer infection time in the trachea, tracheal swabs are more reliable for SARS-CoV-2 detection in these patients.

Pathological findings and patterns of feline infectious peritonitis in the respiratory tract of cats.

Feline infectious peritonitis (FIP) is an important cause of death in cats. Thoracic manifestations are less common than abdominal manifestations, and FIP-associated respiratory disease is poorly documented. This study aimed to investigate pathological findings in the respiratory tract of cats with FIP and the occurrence and distribution of feline coronavirus antigen in the respiratory tract using immunohistochemistry. A retrospective study was carried out on 112 cats with FIP, of which 66 had inflammatory histological lesions in the respiratory tract (58.9%) and were included in this study. Three major gross patterns were defined: marked fibrin deposition in the thoracic cavity with lung atelectasis; marked fibrin deposition in the thoracic cavity with lung pyogranulomas; and lung pyogranulomas without thoracic effusion. Histological analysis revealed primary lesions in the visceral pleura and lung parenchyma at a similar frequency, with multifocal to diffuse presentations. Marked lesions were commonly observed. Five major histological patterns were defined: pleuritis; pleuritis and vasculitis/perivascular injury in the lung parenchyma; pleuritis and pneumonia; perivascular injury in the parenchyma without pleuritis; and pneumonia without pleuritis. In the pleura and pulmonary parenchyma, FIP virus antigen was detected in perivascular and peribronchial macrophages and in macrophages within bronchial-associated lymphoid tissue and foci of necrosis and inflammation in the pleura and lung parenchyma. Co-infections with retroviruses were detected in 47 cats (71.2%), mainly with feline leukemia virus (62.2%). Although FIP is a systemic disease, some cats developed significant lesions in the thoracic cavity, including involvement of the upper respiratory tract and presenting respiratory signs, without other classic signs of FIP. This work advances our knowledge of FIP in the respiratory system, helping veterinarians to recognize the various presentations of this disease.