Airway epithelium respiratory illnesses and allergy (AERIAL) birth cohort: study protocol.

Introduction: Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. Methods: The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to 5 years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to 6 weeks, 1, 3, and 5 years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. Discussion: The AERIAL study will provide a comprehensive longitudinal assessment of factors influencing the association between epithelial dysfunction and respiratory morbidity in early life, and hopefully identify novel targets for diagnosis and early intervention.

An Analysis of the Gut Microbiota and Related Metabolites following PCSK9 Inhibition in Statin-Treated Patients with Elevated Levels of Lipoprotein(a).

BACKGROUND: Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of global mortality, often associated with high blood levels of LDL cholesterol (LDL-c). Medications like statins and PCSK9 inhibitors, are used to manage LDL-c levels and reduce ASCVD risk. Recent findings connect the gut microbiota and its metabolites to ASCVD development. We showed that statins modulate the gut microbiota including the production of microbial metabolites involved in the regulation of cholesterol metabolism such as short chain fatty acids (SCFAs) and bile acids (BAs). Whether this pleiotropic effect of statins is associated with their antimicrobial properties or it is secondary to the modulation of cholesterol metabolism in the host is unknown. In this observational study, we evaluated whether alirocumab, a PCSK9 inhibitor administered subcutaneously, alters the stool-associated microbiota and the profiles of SCFAs and BAs. METHODS: We used stool and plasma collected from patients enrolled in a single-sequence study using alirocumab. Microbial DNA was extracted from stool, and the bacterial component of the gut microbiota profiled following an amplicon sequencing strategy targeting the V3-V4 region of the 16S rRNA gene. Bile acids and SCFAs were profiled and quantified in stool and plasma using mass spectrometry. RESULTS: Treatment with alirocumab did not alter bacterial alpha (Shannon index, p = 0.74) or beta diversity (PERMANOVA, p = 0.89) in feces. Similarly, circulating levels of SCFAs (mean difference (95% confidence interval (CI)), 8.12 [-7.15-23.36] µM, p = 0.25) and BAs (mean difference (95% CI), 0.04 [-0.11-0.19] log10(nmol mg-1 feces), p = 0.56) were equivalent regardless of PCSK9 inhibition. Alirocumab therapy was associated with increased concentration of BAs in feces (mean difference (95% CI), 0.20 [0.05-0.34] log10(nmol mg-1 feces), p = 0.01). CONCLUSION: In statin-treated patients, the use of alirocumab to inhibit PCSK9 leads to elevated levels of fecal BAs without altering the bacterial population of the gut microbiota. The association of alirocumab with increased fecal BA concentration suggests an additional mechanism for the cholesterol-lowering effect of PCSK9 inhibition.

Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) birth cohort: study protocol.

Introduction: Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. Methods and Analysis: The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to five years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to six weeks, one, three, and five years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. Ethics and Dissemination: Ethical approval has been obtained from Ramsey Health Care HREC WA-SA (#1908). Results will be disseminated through open-access peer-reviewed manuscripts, conference presentations, and through different media channels to consumers, ORIGINS families, and the wider community.

Genome Sequences of Two Lytic Staphylococcus aureus Bacteriophages Isolated from Wastewater.

Two lytic double-stranded DNA (dsDNA) bacteriophages, belonging to the family Herelleviridae, were isolated from wastewater in Western Australia. Biyabeda-mokiny 2 appears to belong to the genus Kayvirus, and Koomba-kaat 1 to Silviavirus.

Genome Sequence of a Lytic Staphylococcus aureus Bacteriophage Isolated from Breast Milk.

We identified a double-stranded DNA (dsDNA) bacteriophage appearing to belong to Herelleviridae, genus Kayvirus. The bacteriophage, Biyabeda-mokiny 1, was isolated from breast milk using a clinical isolate of Staphylococcus aureus. The genome is 141,091 bp in length and encodes 230 putative coding sequences.

Complete Genome Sequences of Four Pseudomonas aeruginosa Bacteriophages: Kara-mokiny 8, Kara-mokiny 13, Kara-mokiny 16, and Boorn-mokiny 1.

Pseudomonas aeruginosa is an opportunistic pathogen. Here, we report the isolation of four bacteriophages from wastewater. All four bacteriophages belong to the Myoviridae family. Kara-mokiny 8, 13, and 16 are of the Pbunavirus genus and have genomes between 65,527 and 66,420-bp. Boorn-mokiny 1 is of the Phikzvirus genus and has a 278,796-bp genome.

Complete Genomes of Three Pseudomonas aeruginosa Bacteriophages, Kara-mokiny 1, Kara-mokiny 2, and Kara-mokiny 3.

Here, we present the complete genome sequence of Pseudomonas aeruginosa phages Kara-mokiny 1, Kara-mokiny 2, and Kara-mokiny 3. These phages have lytic capabilities against P. aeruginosa and belong to the myovirus morphotype. The genomes of Kara-mokiny 1 and Kara-mokiny 2 are 67,075 bp while that of Kara-mokiny 3 is 66,019 bp long.

Women in the European Virus Bioinformatics Center.

Viruses are the cause of a considerable burden to human, animal and plant health, while on the other hand playing an important role in regulating entire ecosystems. The power of new sequencing technologies combined with new tools for processing "Big Data" offers unprecedented opportunities to answer fundamental questions in virology. Virologists have an urgent need for virus-specific bioinformatics tools. These developments have led to the formation of the European Virus Bioinformatics Center, a network of experts in virology and bioinformatics who are joining forces to enable extensive exchange and collaboration between these research areas. The EVBC strives to provide talented researchers with a supportive environment free of gender bias, but the gender gap in science, especially in math-intensive fields such as computer science, persists. To bring more talented women into research and keep them there, we need to highlight role models to spark their interest, and we need to ensure that female scientists are not kept at lower levels but are given the opportunity to lead the field. Here we showcase the work of the EVBC and highlight the achievements of some outstanding women experts in virology and viral bioinformatics.

Pseudomonas aeruginosa modulates neutrophil granule exocytosis in an in vitro model of airway infection.

A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air-liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP-10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin-1α and ß were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral-bacterial co-infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV-infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N-formylmethionyl-leucyl-phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.

The bud dormancy disconnect: latent buds of grapevine are dormant during summer despite a high metabolic rate.

Grapevine (Vitis vinifera L.) displays wide plasticity to climate; however, the physiology of dormancy along a seasonal continuum is poorly understood. Here we investigated the apparent disconnect between dormancy and the underlying respiratory physiology and transcriptome of grapevine buds, from bud set in summer to bud burst in spring. The establishment of dormancy in summer was pronounced and reproducible; however, this was coupled with little or no change in physiology, indicated by respiration, hydration, and tissue oxygen tension. The release of dormancy was biphasic; the depth of dormancy declined substantially by mid-autumn, while the subsequent decline towards spring was moderate. Observed changes in physiology failed to explain the first phase of dormancy decline, in particular. Transcriptome data contrasting development from summer through to spring also indicated that dormancy was poorly reflected by metabolic quiescence during summer and autumn. Gene Ontology and enrichment data revealed the prevailing influence of abscisic acid (ABA)-related gene expression during the transition from summer to autumn, and promoter motif analysis suggested that photoperiod may play an important role in regulating ABA functions during the establishment of dormancy. Transcriptomic data from later transitions reinforced the importance of oxidation and hypoxia as physiological cues to regulate the maintenance of quiescence and resumption of growth. Collectively these data reveal a novel disconnect between growth and metabolic quiescence in grapevine following bud set, which requires further experimentation to explain the phenology and dormancy relationships.

Autophagy modulates growth and development in the moss Physcomitrium patens.

Physcomitrium patens apical growing protonemal cells have the singularity that they continue to undergo cell divisions as the plant develops. This feature provides a valuable tool to study autophagy in the context of a multicellular apical growing tissue coupled to development. Herein, we showed that the core autophagy machinery is present in the moss P. patens, and characterized the 2D and 3D growth and development of atg5 and atg7 loss-of-function mutants under optimal and nutrient-deprived conditions. Our results showed that 2D growth of the different morphological and functional protonemata apical growing cells, chloronema and caulonema, is differentially modulated by this process. These differences depend on the protonema cell type and position along the protonemal filament, and growth condition. As a global plant response, the absence of autophagy favors the spread of the colony through protonemata growth at the expense of a reduction of the 3D growth, such as the buds and gametophore development, and thus the adult gametophytic and reproductive phases. Altogether this study provides valuable information suggesting that autophagy has roles during apical growth with differential responses within the cell types of the same tissue and contributes to life cycle progression and thus the growth and development of the 2D and 3D tissues of P. patens.

Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze.

The airway epithelium of children with wheeze is characterized by defective repair that contributes to disease pathobiology. Dysregulation of developmental processes controlled by Notch has been identified in chronic asthma. However, its role in airway epithelial cells of young children with wheeze, particularly during repair, is yet to be determined. We hypothesized that Notch is dysregulated in primary airway epithelial cells (pAEC) of children with wheeze contributing to defective repair. This study investigated transcriptional and protein expression and function of Notch in pAEC isolated from children with and without wheeze. Primary AEC of children with and without wheeze were found to express all known Notch receptors and ligands, although pAEC from children with wheeze expressed significantly lower NOTCH2 (10-fold, p = 0.004) and higher JAG1 (3.5-fold, p = 0.002) mRNA levels. These dysregulations were maintained in vitro and cultures from children with wheeze displayed altered kinetics of both NOTCH2 and JAG1 expression during repair. Following Notch signaling inhibition, pAEC from children without wheeze failed to repair (wound closure rate of 76.9 ± 3.2%). Overexpression of NOTCH2 in pAEC from children with wheeze failed to rescue epithelial repair following wounding. This study illustrates the involvement of the Notch pathway in airway epithelial wound repair in health and disease, where its dysregulation may contribute to asthma development.

Development and validation of a miniaturized bacteriophage host range screening assay against antibiotic resistant Pseudomonas aeruginosa.

Antimicrobial resistance is a current global health crisis, and the increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Prior to clinical application, phage suitability is assessed, via susceptibility testing and breadth of host range to bacteriophage, however, these are both large-scale manual processes and labor-intensive. The aim of the study was to establish and validate a scaled down methodology for high-throughput screening to reduce procedural footprint. In this paper, we describe a scaled-down adapted methodology that can successfully screen bacteriophages, isolated and purified from wastewater samples. Furthermore, we describe a miniaturized host range assay against clinical Pseudomonas aeruginosa isolates using a spot test (2 µL/ drop) that was found to be both sensitive (94.6%) and specific (94.7%). It also demonstrated a positive predictive value (PPV) of 86.4% and negative predictive value (NPV) of 98%. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates was corroborated using the scaled down assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid.

In recent years the study of the commensal microbiota is driving a remarkable paradigm shift in our understanding of human physiology. However, intrinsic technical difficulties associated with investigating the Microbiomics of some body niches are hampering the development of new knowledge. This is particularly the case when investigating the functional role played by the human microbiota in modulating the physiology of key organ systems. A major hurdle in investigating specific Microbiome communities is linked to low bacterial density and susceptibility to bias caused by environmental contamination. To prevent such inaccuracies due to background processing noise, harmonized tools for Microbiomic and bioinformatics practices have been recommended globally. The fact that the impact of this undesirable variability is negatively correlated with the DNA concentration in the sample highlights the necessity to improve existing DNA isolation protocols. In this report, we developed and tested a protocol to more efficiently recover bacterial DNA from low volumes of bronchoalveolar lavage fluid obtained from infants and adults. We have compared the efficiency of the described method with that of a commercially available kit for microbiome analysis in body fluids. We show that this new methodological approach performs better in terms of extraction efficiency. As opposed to commercial kits, the DNA extracts obtained with this new protocol were clearly distinguishable from the negative extraction controls in terms of 16S copy number and Microbiome community profiles. Altogether, we described a cost-efficient protocol that can facilitate microbiome research in low-biomass human niches.

Rhinovirus Infection Drives Complex Host Airway Molecular Responses in Children With Cystic Fibrosis.

Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro. Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF.

The Detection of Bile Acids in the Lungs of Paediatric Cystic Fibrosis Patients Is Associated with Altered Inflammatory Patterns.

Background: Cystic fibrosis (CF) is a hereditary disorder in which persistent unresolved inflammation and recurrent airway infections play major roles in the initiation and progression of the disease. Little is known about triggering factors modulating the transition to chronic microbial infection and inflammation particularly in young children. Cystic fibrosis respiratory disease starts early in life, with the detection of inflammatory markers and infection evident even before respiratory symptoms arise. Thus, identifying factors that dysregulate immune responsiveness at the earliest stages of the disease will provide novel targets for early therapeutic intervention. Methods: We evaluated the clinical significance of bile acid detection in the bronchoalveolar lavage fluid of clinically stable preschool-aged children diagnosed with CF. Results: We applied an unbiased classification strategy to categorize these specimens based on bile acid profiles. We provide clear associations linking the presence of bile acids in the lungs with alterations in the expression of inflammatory markers. Using multiple regression analysis, we also demonstrate that clustering based on bile acid profiles is a meaningful predictor of the progression of structural lung disease. Conclusions: Altogether, our work has identified a clinically relevant host-derived factor that may participate in shaping early events in the aetiology of CF respiratory disease.

Evolutionary insights into FYVE and PHOX effector proteins from the moss Physcomitrella patens.

MAIN CONCLUSION: Genome-wide identification, together with gene expression patterns and promoter region analysis of FYVE and PHOX proteins in Physcomitrella patens, emphasized their importance in regulating mainly developmental processes in P. patens. Phosphatidylinositol 3-phosphate (PtdIns3P) is a signaling phospholipid, which regulates several aspects of plant growth and development, as well as responses to biotic and abiotic stresses. The mechanistic insights underlying PtdIns3P mode of action, specifically through effector proteins have been partially explored in plants, with main focus on Arabidopsis thaliana. In this study, we searched for genes coding for PtdIns3P-binding proteins such as FYVE and PHOX domain-containing sequences from different photosynthetic organisms to gather evolutionary insights on these phosphoinositide binding domains, followed by an in silico characterization of the FYVE and PHOX gene families in the moss Physcomitrella patens. Phylogenetic analysis showed that PpFYVE proteins can be grouped in 7 subclasses, with an additional subclass whose FYVE domain was lost during evolution to higher plants. On the other hand, PpPHOX proteins are classified into 5 subclasses. Expression analyses based on RNAseq data together with the analysis of cis-acting regulatory elements and transcription factor (TF) binding sites in promoter regions suggest the importance of these proteins in regulating stress responses but mainly developmental processes in P. patens. The results provide valuable information and robust candidate genes for future functional analysis aiming to further explore the role of this signaling pathway mainly during growth and development of tip growing cells and during the transition from 2 to 3D growth. These studies would identify ancestral regulatory players undertaken during plant evolution.

Transcriptional Modulation of Polyamine Metabolism in Fruit Species Under Abiotic and Biotic Stress.

Polyamines are growth regulators that have been widely implicated in abiotic and biotic stresses. They are also associated with fruit set, ripening, and regulation of fruit quality-related traits. Modulation of their content confers fruit resilience, with polyamine application generally inhibiting postharvest decay. Changes in the content of free and conjugated polyamines in response to stress are highly dependent on the type of abiotic stress applied or the lifestyle of the pathogen. Recent studies suggest that exogenous application of polyamines or modulation of polyamine content by gene editing can confer tolerance to multiple abiotic and biotic stresses simultaneously. In this review, we explore data on polyamine synthesis and catabolism in fruit related to pre- and postharvest stresses. Studies of mutant plants, priming of stress responses, and treatments with polyamines and polyamine inhibitors indicate that these growth regulators can be manipulated to increase fruit productivity with reduced use of pesticides and therefore, under more sustainable conditions.

Developmental control of hypoxia during bud burst in grapevine.

Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds.