Exertional breathlessness related to medical conditions in middle-aged people: the population-based SCAPIS study of more than 25,000 men and women.

BACKGROUND: Breathlessness is common in the population and can be related to a range of medical conditions. We aimed to evaluate the burden of breathlessness related to different medical conditions in a middle-aged population. METHODS: Cross-sectional analysis of the population-based Swedish CArdioPulmonary bioImage Study of adults aged 50-64 years. Breathlessness (modified Medical Research Council [mMRC] ≥ 2) was evaluated in relation to self-reported symptoms, stress, depression; physician-diagnosed conditions; measured body mass index (BMI), spirometry, venous haemoglobin concentration, coronary artery calcification and stenosis [computer tomography (CT) angiography], and pulmonary emphysema (high-resolution CT). For each condition, the prevalence and breathlessness population attributable fraction (PAF) were calculated, overall and by sex, smoking history, and presence/absence of self-reported cardiorespiratory disease. RESULTS: We included 25,948 people aged 57.5 ± [SD] 4.4; 51% women; 37% former and 12% current smokers; 43% overweight (BMI 25.0-29.9), 21% obese (BMI ≥ 30); 25% with respiratory disease, 14% depression, 9% cardiac disease, and 3% anemia. Breathlessness was present in 3.7%. Medical conditions most strongly related to the breathlessness prevalence were (PAF 95%CI): overweight and obesity (59.6-66.0%), stress (31.6-76.8%), respiratory disease (20.1-37.1%), depression (17.1-26.6%), cardiac disease (6.3-12.7%), anemia (0.8-3.3%), and peripheral arterial disease (0.3-0.8%). Stress was the main factor in women and current smokers. CONCLUSION: Breathlessness mainly relates to overweight/obesity and stress and to a lesser extent to comorbidities like respiratory, depressive, and cardiac disorders among middle-aged people in a high-income setting-supporting the importance of lifestyle interventions to reduce the burden of breathlessness in the population.

New chemical and microbial perspectives on vitamin B1 and vitamer dynamics of a coastal system.

Vitamin B1 (thiamin, B1) is an essential micronutrient for cells, yet intriguingly in aquatic systems most bacterioplankton are unable to synthesize it de novo (auxotrophy), requiring an exogenous source. Cycling of this valuable metabolite in aquatic systems has not been fully investigated and vitamers (B1-related compounds) have only begun to be measured and incorporated into the B1 cycle. Here, we identify potential key producers and consumers of B1 and gain new insights into the dynamics of B1 cycling through measurements of B1 and vitamers (HMP: 4-amino-5-hydroxymethyl-2-methylpyrimidine, HET: 4-methyl-5-thiazoleethanol, FAMP: N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine) in the particulate and dissolved pool in a temperate coastal system. Dissolved B1 was not the primary limiting nutrient for bacterial production and was relatively stable across seasons with concentrations ranging from 74-117 pM, indicating a balance of supply and demand. However, vitamer concentration changed markedly with season as did transcripts related to vitamer salvage and transport suggesting use of vitamers by certain bacterioplankton, e.g. Pelagibacterales. Genomic and transcriptomic analyses showed that up to 78% of the bacterioplankton taxa were B1 auxotrophs. Notably, de novo B1 production was restricted to a few abundant bacterioplankton (e.g. Vulcanococcus, BACL14 (Burkholderiales), Verrucomicrobiales) across seasons. In summer, abundant picocyanobacteria were important putative B1 sources, based on transcriptional activity, leading to an increase in the B1 pool. Our results provide a new dynamic view of the players and processes involved in B1 cycling over time in coastal waters, and identify specific priority populations and processes for future study.

A comprehensive dataset on spatiotemporal variation of microbial plankton communities in the Baltic Sea.

The Baltic Sea is one of the largest brackish water environments on earth and is characterised by pronounced physicochemical gradients and seasonal dynamics. Although the Baltic Sea has a long history of microscopy-based plankton monitoring, DNA-based metabarcoding has so far mainly been limited to individual transect cruises or time-series of single stations. Here we report a dataset covering spatiotemporal variation in prokaryotic and eukaryotic microbial communities and physicochemical parameters. Within 13-months between January 2019 and February 2020, 341 water samples were collected at 22 stations during monthly cruises along the salinity gradient. Both salinity and seasonality are strongly reflected in the data. Since the dataset was generated with both metabarcoding and microscopy-based methods, it provides unique opportunities for both technical and ecological analyses, and is a valuable biodiversity reference for future studies, in the prospect of climate change.

Towards high-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton.

Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we applied a microarray single-cell sequencing (MASC-seq) technology, that generates microscope images of cells in parallel with capturing their transcriptomes, on three species representing important plankton groups with different cell structures; the ciliate Tetrahymena thermophila, the diatom Phaeodactylum tricornutum, and the dinoflagellate Heterocapsa sp. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating "microbial dark matter", although further optimizations are necessary to increase the signal-to-noise ratio.

Optimizing insect metabarcoding using replicated mock communities.

1: Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundances, and biomass. 2: Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization?; and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? 3: We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalizes better across species for homogenates. 4: We conclude that a non-destructive, mild lysis approach shows the highest promise for presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.

Protein N-glycosylation in the bronchoalveolar space differs between never-smokers and long-term smokers with and without COPD.

Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n = 5) and long-term smokers (LTS) with (LTS+, n = 4) and without COPD (LTS-, n = 8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS- and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers.

FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samples.

Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.

Consequences of Using Post- or Prebronchodilator Reference Values in Interpreting Spirometry.

Rationale: Postbronchodilator spirometry is used for the diagnosis of chronic obstructive pulmonary disease. However, prebronchodilator reference values are used for spirometry interpretation. Objectives: To compare the resulting prevalence rates of abnormal spirometry and study the consequences of using pre- or postbronchodilator reference values generated within SCAPIS (Swedish CArdioPulmonary bioImage Study) when interpreting postbronchodilator spirometry in a general population. Methods: SCAPIS reference values for postbronchodilator and prebronchodilator spirometry were based on 10,156 and 1,498 never-smoking, healthy participants, respectively. We studied the associations of abnormal spirometry, defined by using pre- or postbronchodilator reference values, with respiratory burden in the SCAPIS general population (28,851 individuals). Measurements and Main Results: Bronchodilation resulted in higher predicted medians and lower limits of normal (LLNs) for FEV1/FVC ratios. The prevalence of postbronchodilator FEV1/FVC ratio lower than the prebronchodilator LLN was 4.8%, and that of postbronchodilator FEV1/FVC lower than the postbronchodilator LLN was 9.9%, for the general population. An additional 5.1% were identified as having an abnormal postbronchodilator FEV1/FVC ratio, and this group had more respiratory symptoms, emphysema (13.5% vs. 4.1%; P < 0.001), and self-reported physician-diagnosed chronic obstructive pulmonary disease (2.8% vs. 0.5%, P < 0.001) than subjects with a postbronchodilator FEV1/FVC ratio greater than the LLN for both pre- and postbronchodilation. Conclusions: Pre- and postbronchodilator spirometry reference values differ with regard to FEV1/FVC ratio. Use of postbronchodilator reference values doubled the population prevalence of airflow obstruction; this was related to a higher respiratory burden. Using postbronchodilator reference values when interpreting postbronchodilator spirometry might enable the identification of individuals with mild disease and be clinically relevant.

Large-scale phylogenomics of aquatic bacteria reveal molecular mechanisms for adaptation to salinity.

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transitions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive challenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome transitions, resulting in species-level separation between aquatic biomes.

Clinical impact of routine sleep assessment by peripheral arterial tonometry in patients with COPD.

Background: Coexisting obstructive sleep apnoea (OSA) in patients with COPD, defined as overlap syndrome (OVS), is prevalent and underdiagnosed. Routine assessment of OSA is not common practice in COPD care. Our study assessed the clinical impact of sleep assessment by peripheral arterial tonometry (PAT) in COPD patients. Methods: 105 COPD patients (mean age 68.1±9 years, body mass index (BMI) 28.3±6.0 kg·m-2, 44% males, Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I to IV in 2%, 40%, 42% and 16%, respectively) underwent assessment at an outpatient COPD clinic including anthropometrics, arterial blood gas (ABG) and spirometry in this clinical cohort study. PAT-based sleep studies were performed. Predictors of OVS and ABG were determined. Rapid eye movement (REM) sleep-related OSA (REM-OSA) was analysed in OVS. Results: 49 COPD patients (47%) suffered from moderate to severe OSA (OVS group, mean apnoea-hypopnoea index 30.8±18 events·h-1, REM-oxygen desaturation index (REM-ODI) 26.9±17 events·h-1). OVS was more prevalent in males compared to females (59% and 37%, p=0.029, respectively). Age (70.1±8 versus 66.3±10 years), BMI (30.0±6 versus 26.4±7 kg·m-2) and hypertension prevalence (71% versus 45%) were elevated (all p<0.03, respectively), while deep sleep (12.7±7% and 15.4±6%, p=0.029) and mean overnight oxygenation (90.6±3% and 92.3±2%, p=0.003) were lower in OVS compared to COPD alone. REM-ODI was independently associated with daytime arterial carbon dioxide tension (P aCO2 ) (ß=0.022, p<0.001). REM-OSA was associated with an elevated prevalence of atrial fibrillation compared to no REM-OSA (25% and 3%, p=0.022). Conclusions: OVS was highly prevalent, specifically in obese males. REM-related OSA showed strong association with elevated daytime P aCO2 and prevalent cardiovascular disease. PAT was feasible for sleep assessment in COPD.

Linking prokaryotic genome size variation to metabolic potential and environment.

While theories and models have appeared to explain genome size as a result of evolutionary processes, little work has shown that genome sizes carry ecological signatures. Our work delves into the ecological implications of microbial genome size variation in benthic and pelagic habitats across environmental gradients of the brackish Baltic Sea. While depth is significantly associated with genome size in benthic and pelagic brackish metagenomes, salinity is only correlated to genome size in benthic metagenomes. Overall, we confirm that prokaryotic genome sizes in Baltic sediments (3.47 Mbp) are significantly bigger than in the water column (2.96 Mbp). While benthic genomes have a higher number of functions than pelagic genomes, the smallest genomes coded for a higher number of module steps per Mbp for most of the functions irrespective of their environment. Some examples of this functions are amino acid metabolism and central carbohydrate metabolism. However, we observed that nitrogen metabolism was almost absent in pelagic genomes and was mostly present in benthic genomes. Finally, we also show that Bacteria inhabiting Baltic sediments and water column not only differ in taxonomy, but also in their metabolic potential, such as the Wood-Ljungdahl pathway or the presence of different hydrogenases. Our work shows how microbial genome size is linked to abiotic factors in the environment, metabolic potential and taxonomic identity of Bacteria and Archaea within aquatic ecosystems.

ASV portal: an interface to DNA-based biodiversity data in the Living Atlas.

BACKGROUND: The Living Atlas is an open source platform used to collect, visualise and analyse biodiversity data from multiple sources, and serves as the national biodiversity data hub in many countries. Although powerful, the Living Atlas has had limited functionality for species occurrence data derived from DNA sequences. As a step toward integrating this fast-growing data source into the platform, we developed the Amplicon Sequence Variant (ASV) portal: a web interface to sequence-based biodiversity observations in the Living Atlas. RESULTS: The ASV portal allows data providers to submit denoised metabarcoding output to the Living Atlas platform via an intermediary ASV database. It also enables users to search for existing ASVs and associated Living Atlas records using the Basic Local Alignment Search Tool, or via filters on taxonomy and sequencing details. The ASV portal is a Python-Flask/jQuery web interface, implemented as a multi-container docker service, and is an integral part of the Swedish Biodiversity Data Infrastructure. CONCLUSION: The ASV portal is a web interface that effectively integrates biodiversity data derived from DNA sequences into the Living Atlas platform.

Music-based environmental therapy and training programme: a qualitative evaluation study, Norway.

Various authors have emphasized music's value as beneficial intervention, with few or hardly any side effects. Further studies are called for on how music-based environmental treatment in nursing homes works in practice. The aims of the study are first to explore the subjective experiences, opinions and attitudes of health personnel from nursing homes participating in the 'music-based environmental therapy programme (MB programme); and second, to examine why and how this programme impacts on patients and staff, and how it works in practice. It is the first qualitative study to evaluate the impact of the programme on health personnel's daily practice in nursing homes. The sample was strategically selected by means of convenience sampling, and consisted of 26 (n = 26) nurses, managers, physiotherapists, social workers and carers from 11 nursing homes in the south-east of Norway. Data were collected in autumn 2019 using a methodological triangulation of in-depth interviews, focus groups and passive observation, and the data were analyzed using systematic text condensation. With systematic use of music in daily activities in the nursing homes, users became calmer and less outspoken, and the use of psychotropic drugs was greatly reduced. The MB programme seems to be a successful intervention that provides a unique opportunity to improve patients' health and well-being with minimal adverse effects. This new focus on non-pharmacological approaches makes investigation of alternatives to medication vital.

Functional responses of key marine bacteria to environmental change - toward genetic counselling for coastal waters.

Coastal ecosystems deteriorate globally due to human-induced stress factors, like nutrient loading and pollution. Bacteria are critical to marine ecosystems, e.g., by regulating nutrient cycles, synthesizing vitamins, or degrading pollutants, thereby providing essential ecosystem services ultimately affecting economic activities. Yet, until now bacteria are overlooked both as mediators and indicators of ecosystem health, mainly due to methodological limitations in assessing bacterial ecosystem functions. However, these limitations are largely overcome by the advances in molecular biology and bioinformatics methods for characterizing the genetics that underlie functional traits of key bacterial populations - "key" in providing important ecosystem services, being abundant, or by possessing high metabolic rates. It is therefore timely to analyze and define the functional responses of bacteria to human-induced effects on coastal ecosystem health. We posit that categorizing the responses of key marine bacterial populations to changes in environmental conditions through modern microbial oceanography methods will allow establishing the nascent field of genetic counselling for our coastal waters. This requires systematic field studies of linkages between functional traits of key bacterial populations and their ecosystem functions in coastal seas, complemented with systematic experimental analyses of the responses to different stressors. Research and training in environmental management along with dissemination of results and dialogue with societal actors are equally important to ensure the role of bacteria is understood as fundamentally important for coastal ecosystems. Using the responses of microorganisms as a tool to develop genetic counselling for coastal ecosystems can ultimately allow for integrating bacteria as indicators of environmental change.

Chronic rhinosinusitis associated with chronic bronchitis in a five-year follow-up: the Telemark study.

BACKGROUND: Chronic rhinosinusitis (CRS) is associated with generalised airway inflammation. Few studies have addressed the relationship between CRS and chronic bronchitis (CB). METHODS: This prospective study over a five-year period aims to investigate the risk of developing CB in subjects reporting CRS at the beginning of the study. A random sample of 7393 adult subjects from Telemark County, Norway, answered a comprehensive respiratory questionnaire in 2013 and then 5 years later in 2018. Subjects reporting CB in 2013 were excluded from the analyses. New cases of CB in 2018 were analysed in relation to having CRS in 2013 or not. RESULTS: The prevalence of new-onset CB in 2018 in the group that reported CRS in 2013 was 11.8%. There was a significant increase in the odds of having CB in 2018 in subjects who reported CRS in 2013 (OR 3.8, 95% CI 2.65-5.40), adjusted for age, sex, BMI, smoking and asthma. CONCLUSION: In this large population sample, CRS was associated with increased odds of developing CB during a five-year follow-up. Physicians should be aware of chronic bronchitis in patients with CRS.

Development of a Digital Tool for People With a Long-Term Condition Using Stroke as a Case Example: Participatory Design Approach.

BACKGROUND: In patient care, demand is growing for digital health tools to enable remote services and enhance patient involvement. People with chronic conditions often have multiple health problems, and long-term follow-up is recommended to meet their needs and enable access to appropriate support. A digital tool for previsit preparation could enhance time efficiency and guide the conversation during the visit toward the patient's priorities. OBJECTIVE: This study aims to develop a digital previsit tool and explore potential end user's perceptions, using a participatory approach with stroke as a case example. METHODS: The digital tool was developed and prototyped according to service design principles, informed by qualitative participant data and feedback from an expert panel. All features were processed in workshops with a team that included a patient partner. The resulting tool presented questions about health problems and health information. Study participants were people with stroke recruited from an outpatient clinic and patient organizations in Sweden. Development and data collection were conducted in parallel. For conceptualization, the initial prototype was based on the Post-Stroke Checklist and research. Needs and relevance were explored in focus groups, and we used a web survey and individual interviews to explore perceived utility, ease of use, and acceptance. Data were thematically analyzed following the Framework Method. RESULTS: The development process included 22 participants (9 women) with a median age of 59 (range 42-83) years and a median of 51 (range 4-228) months since stroke. Participants were satisfied or very satisfied with using the tool and recommended its use in clinical practice. Three main themes were constructed based on focus group data (n=12) and interviews (n=10). First, valuable accessible information illuminated the need for information to confirm experiences, facilitate responses, and invite engagement in their care. Amendments to the information in turn reconfigured their expectations. Second, utility and complexity in answering confirmed that the questions were relevant and comprehensible. Some participants perceived the answer options as limiting and suggested additional space for free text. Third, capturing needs and value of the tool highlighted the tool's potential to identify health problems and the importance of encouraging further dialog. The resulting digital tool, Strokehälsa [Strokehealth] version 1.0, is now incorporated into a national health platform. CONCLUSIONS: The participatory approach to tool development yielded a previsit digital tool that the study group perceived as useful. The holistic development process used here, which integrated health information, validated questions, and digital functionality, offers an example that could be applicable in the context of other long-term conditions. Beyond its potential to identify care needs, the tool offers information that confirms experiences and supports answering the questions in the tool. The tool is freely shared for adaptation in different contexts. TRIAL REGISTRATION: researchweb 236341; https://www.researchweb.org/is/vgr/project/236341.

Glucose Homeostasis in Relation to Neutrophil Mobilization in Smokers with COPD.

Purpose: Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS) are common comorbidities in chronic obstructive pulmonary disease (COPD), but the underlying pathogenic mechanisms are poorly understood. Given that these morbidities all display increased neutrophil mobilization, the current study aimed to address whether glucose homeostasis relates to signs of neutrophil mobilization in COPD. Methods: The study population included healthy non-smokers (HNS) and long-term smokers without (LTS) and with COPD (LTS+COPD). No subject had T2DM or MetS. Serum cotinine was quantified to evaluate current smoking. Capillary blood glucose was measured after overnight fasting and during an oral glucose tolerance test (OGTT). Neutrophils were quantified in blood and bronchoalveolar lavage samples (BAL). The neutrophil-related cytokines IL-36α, -ß and -γ were quantified (ELISA) along with IL-6, IL-8, INF-γ and CXCL10 (U-Plex®) in plasma and cell-free BAL fluid (BALF). In addition, we quantified neutrophil elastase (ELISA) and net proteinase activity (substrate assay) in BALF. Results: The LTS+COPD group had lower fasting glucose, greater change in glucose during OGTT and higher neutrophil concentrations in BAL and blood compared with HNS. Fasting glucose correlated in a positive manner with blood neutrophil concentration, forced expiratory volume in 1 second/forced vital capacity ratio (FEV1/FVC) and FEV1 (% of predicted) in LTS+COPD. In this group, the concentration of IL-36α in BALF correlated in a negative manner with fasting glucose, blood neutrophil concentration and FEV1, while the CXCL10 concentration in BALF correlated in a negative manner with glucose at the end of OGTT (120 min). We observed no corresponding correlations for neutrophil elastase, net proteinase or gelatinase activity. Conclusion: In smokers with COPD, altered glucose homeostasis is associated with local and systemic signs of increased neutrophil mobilization, but not with local proteinases. This suggests that other specific aspects of neutrophil mobilization constitute pathogenic factors that affect glucose homeostasis in COPD.

Evaluating metagenomic assembly approaches for biome-specific gene catalogues.

BACKGROUND: For many environments, biome-specific microbial gene catalogues are being recovered using shotgun metagenomics followed by assembly and gene calling on the assembled contigs. The assembly is typically conducted either by individually assembling each sample or by co-assembling reads from all the samples. The co-assembly approach can potentially recover genes that display too low abundance to be assembled from individual samples. On the other hand, combining samples increases the risk of mixing data from closely related strains, which can hamper the assembly process. In this respect, assembly on individual samples followed by clustering of (near) identical genes is preferable. Thus, both approaches have potential pros and cons, but it remains to be evaluated which assembly strategy is most effective. Here, we have evaluated three assembly strategies for generating gene catalogues from metagenomes using a dataset of 124 samples from the Baltic Sea: (1) assembly on individual samples followed by clustering of the resulting genes, (2) co-assembly on all samples, and (3) mix assembly, combining individual and co-assembly. RESULTS: The mix-assembly approach resulted in a more extensive nonredundant gene set than the other approaches and with more genes predicted to be complete and that could be functionally annotated. The mix assembly consists of 67 million genes (Baltic Sea gene set, BAGS) that have been functionally and taxonomically annotated. The majority of the BAGS genes are dissimilar (< 95% amino acid identity) to the Tara Oceans gene dataset, and hence, BAGS represents a valuable resource for brackish water research. CONCLUSION: The mix-assembly approach represents a feasible approach to increase the information obtained from metagenomic samples. Video abstract.

Salivary Microbiota and Host-Inflammatory Responses in Periodontitis Affected Individuals With and Without Rheumatoid Arthritis.

Objectives: Periodontitis and rheumatoid arthritis (RA) are two widespread chronic inflammatory diseases with a previously suggested association. The objective of the current study was to compare the oral microbial composition and host´s inflammatory mediator profile of saliva samples obtained from subjects with periodontitis, with and without RA, as well as to predict biomarkers, of bacterial pathogens and/or inflammatory mediators, for classification of samples associated with periodontitis and RA. Methods: Salivary samples were obtained from 53 patients with periodontitis and RA and 48 non-RA with chronic periodontitis. The microbial composition was identified using 16S rRNA gene sequencing and compared across periodontitis patients with and without RA. Levels of inflammatory mediators were determined using a multiplex bead assay, compared between the groups and correlated to the microbial profile. The achieved data was analysed using PCoA, DESeq2 and two machine learning algorithms, OPLS-DA and sPLS-DA. Results: Differential abundance DESeq2 analyses showed that the four most highly enriched (log2 FC >20) amplicon sequence variants (ASVs) in the non-RA periodontitis group included Alloprevotella sp., Prevotella sp., Haemophilus sp., and Actinomyces sp. whereas Granulicatella sp., Veillonella sp., Megasphaera sp., and Fusobacterium nucleatum were the most highly enriched ASVs (log2 FC >20) in the RA group. OPLS-DA with log2 FC analyses demonstrated that the top ASVs with the highest importance included Vampirovibrio sp. having a positive correlation with non-RA group, and seven ASVs belonging to Sphingomonas insulae, Sphingobium sp., Novosphingobium aromaticivorans, Delftia acidovorans, Aquabacterium spp. and Sphingomonas echinoides with a positive correlation with RA group. Among the detected inflammatory mediators in saliva samples, TWEAK/TNFSF12, IL-35, IFN-α2, pentraxin-3, gp130/sIL6Rb, sIL-6Ra, IL-19 and sTNF-R1 were found to be significantly increased in patients with periodontitis and RA compared to non-RA group with periodontitis. Moreover, correlations between ASVs and inflammatory mediators using sPLS-DA analysis revealed that TWEAK/TNFSF12, pentraxin-3 and IL-19 were positively correlated with the ASVs Sphingobium sp., Acidovorax delafieldii, Novosphingobium sp., and Aquabacterium sp. Conclusion: Our results suggest that the combination of microbes and host inflammatory mediators could be more efficient to be used as a predictable biomarker associated with periodontitis and RA, as compared to microbes and inflammatory mediators alone.

Short- and long-read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.