Les oeufs de poules en liberté et les oeufs provenant « d’élevage confort » sont maintenant des produits communs dans les marchés alimentaires du Québec. Cette nouvelle variété de produits est le résultat de l’évolution des pratiques afin de concilier le bien-être des poules pondeuses et les besoins de l’industrie. En effet, d’ici l’année 2036, les logements conventionnels pour poules pondeuses (logements grillagés pour des petits groupes de poules) seront graduellement remplacés par des logements alternatifs. Les logements alternatifs pour poules pondeuses regroupent les colonies enrichies et les logements sans cages (sur plancher ou en volière). Ils ont en commun une plus grande surface et un plus grand espace vertical pour les animaux ainsi que la présence d’enrichissements, tels que des perchoirs et des nids reclus, qui permettent aux poules d’exprimer davantage leurs comportements naturels. Les logements sans cages se démarquent des colonies enrichies par la présence de litière au sol, qui est généralement composée de copeaux de bois ou de matériel sablonneux, sur laquelle les poules peuvent picorer, gratter et prendre des bains de sable. L’adoption des logements alternatifs pour poules pondeuses impliquera de nombreux changements d’infrastructures et de pratiques de travail. La plus grande liberté de mouvement des animaux et la présence de surface de litière dans certains poulaillers pourraient entraîner de plus hautes concentrations de poussières et de particules biologiques dans l’air de ces environnements de travail. Toutefois, la qualité de l’air des poulaillers alternatifs au Québec n’a pas encore été caractérisée, même si un effet sur la santé respiratoire des travailleurs et des travailleuses de l’industrie ovocole est à prévoir. Ce projet a permis d’évaluer les concentrations de poussières, de bactéries et de moisissures en poulaillers conventionnels, en colonies enrichies et en logements sans cages de type volière au Québec. De plus hautes concentrations de poussières, d’endotoxines et de certaines bactéries ont été trouvées dans les volières. Toutefois, le type de poulailler n’a pas eu le même effet pour d’autres bioaérosols. La diversité bactérienne dans l’air des volières se distingue de celle trouvée dans les poulaillers utilisant des logements grillagés, et est également similaire à celle retrouvée dans la litière. Un suivi longitudinal des concentrations de bactéries totales et d’archées totales en poulaillers alternatifs a révélé l’effet de la saison sur les concentrations des bioaérosols à l’étude. Compte tenu des plus hautes concentrations de poussières dans les volières, un essai en poulaillers expérimentaux à petite échelle a révélé le potentiel de méthodes de mitigation des poussières et de l’ammoniac sur les bioaérosols, notamment l’aspersion d’émulsion d’huile sur la surface de litière. Un essai en volière commerciale a ensuite confirmé l’importance du dosage et de la fréquence d’aspersion pour réduire les poussières et l’ammoniac. Enfin, des données préliminaires quant à la santé respiratoire des travailleurs et des travailleuses montre la pertinence de poursuivre et de raffiner l’évaluation de l’effet des nouvelles pratiques sur la santé humaine par le recrutement d’une plus importante cohorte.

Mechanically ventilated livestock operations continuously emit bioaerosols containing bacteria and antimicrobial resistance genes (ARGs), yet their environmental dispersion remains poorly characterized. This study assessed the characterization of airborne microbial and ARG markers from two pig finishing buildings (PFBs) and two broiler chicken barns (BCBs) across a one-kilometer distance gradient. Air samples were analyzed for microbial and ARG concentrations and bacterial diversity. A one-phase exponential decay model revealed a rapid decline in airborne concentrations within the first 10 meters from the source, with half-distances generally below 10 meters. Despite differences in animal species and husbandry conditions, reduction rates were similar across farm types, suggesting consistent physical dispersion processes. Microbial diversity indices did not vary significantly with distance, but the relative abundance of bacterial genera showed shifts in community structures along the distance gradient. Bacteria profiles around farms were different from those compared to control sites. Network analyses revealed denser ARG-bacteria associations in PFBs, particularly involving tetracyclines and macrolides resistances, compared to BCBs. These findings suggest that emitted bioaerosols can persist beyond immediate farm boundaries and contribute to background environmental levels. This study highlights the importance of including air in surveillance programs and supports the need for integrated One Health approaches to better understand and mitigate the environmental spread of pathogens and antimicrobial resistance.

BACKGROUND AND AIMS:Antibiotics are natural compounds produced by microorganisms that have long existed in ecosystems. However, the widespread clinical and agricultural use of antibiotics has intensified selective pressures on bacteria, leading to the proliferation of antibiotic resistance genes (ARGs). The increasing prevalence of these genetic elements in clinical and environmental settings now poses a major global health threat. While ARGs are well documented in anthropogenically influenced environments, their distribution and origins in remote ecosystems, such as boreal forests, remain poorly understood. Here, we investigate the occurrence, diversity and potential origins of ARGs in the boreal lichen Cladonia stellaris. METHODS:We conducted the first targeted assessment of ARGs in lichens by analysing 42 C. stellaris samples from northern and southern lichen woodlands in eastern Canada. Using high-throughput quantitative PCR, we screened for 33 ARGs and three mobile genetic elements (MGEs), quantifying their relative abundance. Bacterial community composition was characterized via 16S rRNA gene sequencing. Statistical analyses evaluated geographical patterns, co-occurrence between ARGs and bacterial taxa, and the influence of latitude on ARG distribution. KEY RESULTS:Ten ARGs conferring resistance to four antibiotic classes (aminoglycosides, beta-lactams, quinolones and sulfonamides), along with one MGE, were detected. The ARGs blaCTX-M-1, qnrB and qepA were highly prevalent, with qepA often surpassing 16S rRNA gene abundance. Only qnrB showed significantly higher abundance in southern samples. Latitude significantly influenced ARG profiles, whereas bacterial community composition did not. CONCLUSIONS:Our findings demonstrate that C. stellaris harbours diverse ARGs in remote boreal ecosystems with limited anthropogenic influence. Proposed explanations for ARG presence include long-distance dispersal via bioaerosols and endogenous development within lichen microbiomes, yet these remain speculative. Future work incorporating bacterial isolation, whole-genome sequencing, metatranscriptomics, air sampling and metabolomic profiling is necessary to unravel the ecology and evolution of ARGs in natural habitats.

Indoor air contamination and fungal growth in buildings are important factors influencing indoor environmental quality. This study assessed airborne and surface-associated fungi in 60 dwellings in Nunavik across summer 2023 and winter 2024 using an activated air sampling protocol with the SASS (R) 3100 Dry Air Sampler (300 L/min), the first such application in a remote northern context. Airborne fungal concentrations showed strong seasonal variation. Cladosporium peaked in summer (10(3) copies/m(3)) but was nearly absent in winter, while Penicillium/Aspergillus declined from 10(4) to 10(3) copies/m(3). Water damage-associated (C. globosum and T. viride) and human health-relevant species (A. fumigatus and A. versicolor) decreased by >2 logs from summer to winter. S. chartarum remained consistent across seasons (10(1)-10(2) copies/m(3)). Outdoor sampling confirmed contributions from Penicillium and Aspergillus, whereas water damage moulds were mostly absent outdoors, supporting their value as indoor moisture contamination markers. Surface sampling revealed diverse fungal communities across both seasons, dominated by Aspergillus, Penicillium, and Cladosporium, with the highest diversity in bathrooms. Yeasts such as Rhodotorula and Cryptococcus were persistently isolated, reflecting occupant contributions. We also demonstrated that activated air sampling is particularly valuable for detecting moulds that are not apparent through surface inspection. Overall, these findings highlight pronounced seasonal dynamics in indoor fungal loads, the influence of outdoor air and ventilation, and the importance of monitoring water damage-associated moulds as indicators of indoor contamination in northern housing for establishing baseline data for future assessments

While indoor microbiota have been increasingly studied, microbial communities in subarctic and Arctic Canadian housing remain poorly characterized. In this sub-study, we did a methodological comparison of microbial community composition across different dust-based matrices (indoor settled dust, heating and ventilation system filter dust, and activated air) in six Inuit homes of Nunavik, Quebec, Canada. Activated air sampling, which involves resuspension of settled dust, remains underexplored in indoor microbiota research and has rarely been compared directly with dust-based matrices using high-throughput sequencing. Fungal biomass was assessed with quantitative PCR (qPCR) targeting the indoor fungal markers Penicillium and Aspergillus spp., as well as the outdoor marker Cladosporium spp. The Environmental Relative Moldiness Index (ERMI) metric was used to assess fungal contamination, and microbial communities were described using high-throughput amplicon-based sequencing of the ITS2 and 16S rRNA genes. Activated air samples exhibited relatively low biomass, preventing the calculation of the ERMI metric. However, house dust and heating and ventilation system filter dust showed similarities in terms of ERMI-species detected. ITS2 amplicon-based sequencing revealed broadly similar fungal communities across the three matrices, whereas 16S sequencing showed that bacterial communities differed more strongly, with activated air enriched in human-associated and transient taxa. This sub-study provides one of the first integrated comparisons of activated air and other dust-based matrices combining ERMI-like assessment with fungal and bacterial high-throughput sequencing. This work highlights the strengths and limitations of dust-based sampling methods and supports the selection of appropriate methodological strategies for indoor microbiota assessment in remote northern housing.

Barrier face coverings were commonly used during the SARS-CoV-2 pandemic as a solution for the shortage of medical-grade masks. Many of these devices are designed to be washable and reusable. As concerns rise about the environmental repercussions of single-use masks, the use of efficient, reusable barrier face coverings has begun to emerge as an alternative. If the ASTM standard F3502-21 governs the efficiency of inert particle filtration, it does not, however, require that devices be tested against viral aerosols. Although the impact of repeated washes on these devices' ability to filter inert particles has been investigated, disagreements remain on whether washing barrier face coverings can significantly affect their efficiency. The goal of this study was to evaluate the impact of washes on the filtration efficiency of five barrier face coverings from a single manufacturer against submicron particles and a model virus. To that end, a wind tunnel specifically designed for the evaluation of masks' filtration efficiency was used in conjunction with bacteriophage PhiX174, which proxied for human pathogens. The five barrier face coverings were washed and tested at different washing frequencies, determined as per the manufacturer's indications. Results showed a significant loss of efficiency in filtering submicron particles and viruses for all five barrier face coverings. Resistance to washing varied across the tested devices. Reusable barrier face coverings that are intended to substitute for medical-grade masks should be tested after undergoing a maximum number of washes to provide an accurate assessment of their filtration efficiency at the time of use.Copyright (c) 2025 American Association for Aerosol Research

Background: Pembrolizumab is a monoclonal antibody (mAb) approved for treating Non-Small Cell Lung Cancer (NSCLC), melanoma and lymphomas. Commercialized in single-size (100 mg/4 mL) vials, the pembrolizumab solution contains no preservative. As such, the manufacturer recommends using pembrolizumab vials only once, and thus, to rapidly dispose of any unused portion. Thus, appreciable amounts of this costly product are wasted. Objective: To evaluate the physical, chemical and microbiological stability of pembrolizumab vial leftovers stored at room temperature or at 4 °C, 7 and 14 days after first vial puncturing. Methods: Following pH assessments, submicronic aggregation and turbidity of pembrolizumab were measured by dynamic light scattering (DLS) and spectrophotometry, respectively. In addition, SE-HPLC (size-exclusion high-performance liquid chromatography), IEX-HPLC (ion exchange HPLC) and peptide mapping HPLC served to respectively evaluate aggregation and fragmentation, distribution of charge and primary structure of pembrolizumab. Incubation at 37 °C for 48 h of pembrolizumab vial leftovers on blood agar plates was used to determine their microbiological stability. Results: Physical, chemical and microbiological stability of pembrolizumab leftovers was demonstrated for at least two full weeks. Conclusions: These results argue forcefully in favor of allowing prolongation of pembrolizumab vial leftovers usage well beyond a single day.

The large-scale abundance and distribution of antibiotic resistance genes (ARGs) within the atmosphere remains poorly documented, particularly over oceans. This study explores bacterial loads, diversity, and associated antimicrobial resistance genes in aerosols over the North Atlantic Ocean. Aerosol samples were collected from a ship during a cruise from Brest (France) to Woods Hole (USA) for 24-h periods using a mast-mounted system, with additional one-hour spot samples taken daily and nightly using high-flow rates samplers. The airborne concentrations of bacteria along with 21 ARG subtypes as indicators of key resistance families were monitored using qPCR. These were related to the bacterial diversity obtained from same samples through ribosomal gene amplicon sequencing, and to the geographical origin of the air masses estimated using atmospheric dynamics models. Total ARG concentrations ranged from background concentrations of a few copies to >105 copies/m3 of air. Near coasts, macrolide and tetracycline resistance genes were dominant (up to 93 % and 38 % of the total ARG monitored here, respectively). While sulfonamide resistance genes were also detected further offshore, those related to transposases and β-lactamases were detected only sporadically. The multiple observed correlations between the aforementioned gene concentrations in the air and potential soil-derived microorganisms may be indicative of continental inputs. Conversely, the prevalence of quinolone resistance (qepA) in the air over the open ocean points toward a contribution from marine surfaces, supported by associations between several ARGs and marine microorganisms including cyanobacteria. These may thus act as environmental reservoirs of ARGs, and sources for further environmental spread notably by air means.

Highlights Fan exhaust air sampling is a reliable monitoring proxy for indoor bioaerosols from livestock operations. Air samples collected indoors and at fan exhaust have highly similar bacterial diversity. At low indoor concentrations, specific microbial markers are still detectable in the air collected at the fan exhaust. Abstract. The incidence of animal and zoonotic diseases is expected to increase in the coming years, imposing the reinforcement of biosecurity measures for livestock operations. Airborne transmission of certain infectious agents underscores the importance of surveilling bioaerosols. However, having access to livestock operations for monitoring purposes is now challenging. Hence, it has become imperative to explore alternative strategies to assess indoor bioaerosols. This study aimed to compare bacterial diversity and quantify microbial markers found in bioaerosols indoors and at the fan exhausts of pig-finishing buildings (PFBs) and broiler chicken barns (BCBs). Bioaerosols were collected using a filter-based, high-flow rate air sampler in 12 facilities (10 PFBs and 2 BCBs) during the warm season in Eastern Canada, corresponding to maximal ventilation rate operations. Four farms—PFB-1, PFB-2, BCB-1, and BCB-2—were visited multiple times, while the other eight PFBs (PFB-3 to PFB-10) were visited once. At each farm, indoor air samples were paired with samples from the corresponding sidewall extraction fans. Amplicon-based sequencing and quantitative PCR (qPCR) were performed to describe bacterial diversity and quantify specific microbial (bacterial and archaeal 16S rRNA genes, Enterococcus spp., and a phage of Aerococcus viridans) and animal (swine and poultry DNA) markers. No significant differences in OTUs abundance and diversity between indoor bioaerosols and their corresponding fan exhaust samples were observed. There were also no significant differences between an indoor and its corresponding fan exhaust air sample when comparing OTUs relative abundance and their presence-absence. Similarly, concentrations of bacterial 16S rRNA genes in indoor samples (10 6 –10 8 ) did not significantly differ from those found in samples collected at the fan exhaust (10 5 –10 8 ) for both PFBs and BCBs. Strong correlations were observed between sampling sites for Archaea, Enterococcus, and A. viridans phage concentrations while poultry and swine DNA concentrations at fan exhausts did not correlate with indoor levels. All investigated markers were detectable at fan exhausts, even at low indoor concentrations (10 2 –10 3 ). Our study suggests that air sampling at the fan exhaust of barns provides a representative picture of the indoor bioaerosols both for bacterial diversity and barn-specific indicators when the fans are in use. This method appears promising for characterizing indoor air quality based on emissions and could be highly valuable in cases where biosecurity measures or outbreaks restrict access to barns. Keywords: Air sampling, Airborne microbiota, Bioaerosols, Broiler, Fan exhaust, Livestock operations, Pig.

BACKGROUND:The exhaled breath of infected, mechanically ventilated patients poses an infection risk to health care workers. Proper expiratory gas filtration with heat and moisture exchanger filters (HMEF) or filters could reduce that phenomenon. Current laboratory means of assessing the filtration efficiency are limited to the use of monodisperse aerosols at a single humidity level and flow. This study aims to examine the filtration efficiency of various devices under simulated clinical conditions, namely against a broad range of particle sizes containing viruses at different levels of gas humidity and flow. METHODS:A wind tunnel was adapted to evaluate the filtration efficiency of 4 devices (HME, HMEF, filters, and HEPA-HMEF) against viral aerosols. Bacteriophages PhiX174 and MS2 were used as a proxy for human viruses. RESULTS:In general, particulate filtration was significantly increased under dry versus humid conditions and with low versus high flows (P < .05). The HEPA filter significantly outperformed all other devices under all tested conditions in filtration efficiency. Both HMEF and filter showed approximately a 1% decrease in absolute differences compared with the reference method (∼99% vs 99.99%). This difference could represent an emission of as many as 102 SARS-CoV-2 copies per hour by an ICU patient, which is enough to spread the infection. CONCLUSIONS:Accurate testing of filtration function has long gone unexamined, and in preparation for the next respiratory pandemic, better evaluation of devices that filter potentially dangerous pathogens is vital for health care professionals and systems. Standard filtration testing should be adapted to mimic the clinical usage of HMEs and filters.

The COVID-19 pandemic brought global attention to indoor air quality (IAQ), which increases public’s awareness on monitoring indoor ventilation conditions significantly. Indoor CO2 monitoring has been widely accepted as an effective way for indicating IAQ conditions, attributed to its close relationships with indoor air change rates. However, real-time estimation of air change rates or CO2 emission rates from CO2 measurement data remains challenging due to uncertainties in factors like random air movements, dynamic conditions (e.g., weather and occupancy), and the limitations of deterministic equations. This study addresses these challenges by applying Bayesian inference to a stochastic CO2-based grey-box model, enabling the accurate estimation of ventilation and CO2 emission rates while accounting for uncertainty. The model’s accuracy and robustness were validated through CO2 tracer gas experiments, employing constant injection and decay methods in an airtight chamber. Both prior and posterior predictive checks (PPC) were performed to verify this approach. The approach proposed by this study improves the interpretation of CO2 monitoring data, thereby facilitating the future real-time IAQ management.

Total outward leakage reduction efficiency against viral particles for masks is not well known. The objective of this study was to evaluate this efficiency for various masks against virus-containing polydisperse aerosols. To achieve that, a test bench previously built for particulate total outward leakage reduction efficiency evaluation was adapted to generate viral aerosols. Total outward leakage reduction efficiency against viral particles was measured for 10 different masks using a wind tunnel and a mannequin head. The impact of washing on the total outward leakage reduction efficiency was assessed for three barrier face coverings against viral particles. Total outward leakage reduction efficiency for viruses was generally higher than for particles (0.52-3.3 mu m) since each of these particles could contain more than one virion and have a greater impact on the viral efficiency compared to particles. Washing did not have a major impact on the efficiency measured for the barrier face coverings tested. Total outward leakage reduction efficiency tests could be done using inert particles since total outward leakage reduction efficiency was lower against particles (0.52-3.3 mu m) than against infectious viruses. However, using biological particles may be a better way to interpret the risk associated with infectious aerosols.Copyright (c) 2025 American Association for Aerosol Research

[This corrects the article DOI: 10.1371/journal.pone.0231164.].

Wastewater is a known carrier for human pathogenic viruses, with seasonal variations in concentrations, and wastewater treatment plant (WWTP) workers are a potentially overlooked occupational group regarding exposure to secondary aerosolized viruses. Exposure assessment of airborne pathogens is complicated by a lack of universal markers of viruses, no standardized sampling protocol, and challenges in detecting extremely low-abundant targets. In this study, we evaluate the risk of workers' exposure to 4 pathogens, Adenovirus, Norovirus GI and GII, and Influenza A and the Pepper mild mottle virus (PMMoV) as an indicator for aerosolized viruses from wastewater, in 3 WWTPs in the Oslo region, Norway. We collected personal and stationary air samples in summer and winter and used digital droplet PCR (ddPCR) to enable the detection of low-abundant targets. Pathogenic viruses were detected in 22% of all samples, with similar detection rates in personal and stationary samples, with a maximum concentration of 762 genome copies/m3 air. PMMoV was detected in 69% of all samples, with concentrations ranging from 28 to 9703 genome copies/m3 air. The pathogens and PMMoV were most frequently detected at the grids, biological cleansing, sedimentation basins, and sludge treatment/de-watering stations, and were associated with tasks such as flushing, cleaning, and maintenance of the same workstations. Overall, the concentration of pathogens and PMMoV in the air was low, but there is a potential for high point exposure which may pose a risk to workers' health and is increased by the nature of the workers' tasks. PMMoV may be a promising tool for assessing the overall potential for viruses with human waste origin aerosolized from sewage. To strengthen this indicator-based approach to occupational exposure assessment, we recommend validating PMMoV along with other potential indicators. Validation should include evaluating the correlation between these indicators and pathogens in both wastewater and bioaerosols.

BACKGROUND:The SARS-CoV-2 Omicron variant is transmitted via contaminated droplets and aerosols, raising concerns in healthcare settings where poor ventilation and high patient density can increase airborne viral load. AIM:This study aimed to assess real-world exposure of healthcare workers to COVID-19-positive patients isolated in designated hospital areas, using continuous 24-h air sampling. METHODS:Air sampling was conducted inside 10 hospital rooms hosting a succession of 38 patients who tested positive for SARS-CoV-2. Sampling was performed using 37-mm cassettes placed near the patients' heads. The Omicron variant in the air was detected by RT-qPCR, with results expressed as emission rates based on air changes per hour for each room and correlated with the onset of patients' symptoms. FINDINGS:The SARS-CoV-2 was detected and quantified in the air of 89% of patients, indicating that 76.7% of the rooms hosting positive patients had detectable levels of airborne virus. This corresponded to an average viral emission rate of 1.45 × 105 ± 2.16 × 105 genomes/h per patient. Expectoration was the sole symptom significantly affecting emission rates, with patient suffering from it exhibiting values three times higher than patients without. Additionally, the room accounted for half of the variance in emission rates, suggesting that the number of patients and the room's prior usage are key determinants of viral particle exposure. CONCLUSION:Our findings indicate that healthcare workers face significant exposure when providing care in rooms with positive patients, even when mechanically ventilated. Greater attention should be given to treating and managing these spaces to reduce the potential for viral transmission toward healthcare workers.

During the SARS-CoV-2 pandemic, masks were widely used to reduce the spread of the virus through aerosols and droplets. While these are generally tested in laboratories for their effectiveness against particles and bacteria, their efficiency against viruses is seldom evaluated. Given the absence of standardized rules governing filtration efficiency against viruses, this study sought to examine how various types of masks perform against virus-containing polydisperse aerosols. Additionally, it aimed at assessing the consistency of mask filtration performances under similar test conditions, considering the heterogeneous nature of the standards for particles. Masks' filtration efficiencies were determined using a wind tunnel specially designed for this kind of testing. Bacteriophages were used as a proxy for human viruses. Overall, the viral filtration efficiency was higher than that of particles. No significant difference was observed between infectious and total viruses. Particulate filtration performance varied among masks compared to their standard requirements. Filtration efficiency testing should report the specific size used whether it was tested with mono- or polydisperse aerosols to gain a clearer understanding of their effectiveness.

In the original publication [...].

BACKGROUND:Hospital-associated infections caused by carbapenemase-producing organisms (CPOs) pose a significant health concern. Healthcare settings implement measures to control the spread of CPOs and prevent outbreaks, but the role of air in disseminating carbapenemase genes remains unclear. This study assessed three carbapenemase-associated genes (blaKPC, blaOXA-48 and blaNDM) in the environment of CPO-colonized patients. METHODS:A prospective observational study was conducted in four hospitals in Quebec, Canada in the rooms of CPO-colonized patients. Air was collected actively inside the rooms of CPO-colonized patients, and floor and no-touch surfaces were sampled using pre-moistened swabs and sponges; the findings were compared with those from control rooms (i.e. rooms hosting non-CPO-colonized patients) located on the same floor. Additional floor samples were collected in adjacent hallways to estimate potential dissemination within the settings. The presence and abundance of carbapenemase-producing genes (blaKPC, blaNDM and blaOXA-48) were evaluated using quantitative polymerase chain reaction. RESULTS:Carbapenemase-encoding genes were detected frequently in CPO-colonized patient environments, notably on floors (97% of detection frequency), door frames (52%), and no-touch surfaces (42%). Conversely, only one air sample tested positive for blaKPC. These genes were also detected in hallways adjacent to the rooms of CPO-colonized patients (92%), control rooms (100%), and hallways adjacent to the rooms of non-CPO-colonized patients (78%), with abundance decreasing with distance from CPO-colonized rooms. CONCLUSION:These findings suggest that carbapenem resistance can spread within healthcare settings, and air may play a role in gene dissemination. Additional measures should be considered to limit resistance gene transfer, particularly via floors and air.