Implementation of Novel Affinity Ligand for Lentiviral Vector Purification.

The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation.

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP's therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG's anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1ß, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.

Chemical characterisation of particulate matter in urban transport modes.

Traffic is a main source of air pollutants in urban areas and consequently daily peak exposures tend to occur during commuting. Personal exposure to particulate matter (PM) was monitored while cycling and travelling by bus, car and metro along an assigned route in Lisbon (Portugal), focusing on PM2.5 and PM10 (PM with aerodynamic diameter <2.5 and 10 µm, respectively) mass concentrations and their chemical composition. In vehicles, the indoor-outdoor interplay was also evaluated. The PM2.5 mean concentrations were 28 ± 5, 31 ± 9, 34 ± 9 and 38 ± 21 µg/m3 for bus, bicycle, car and metro modes, respectively. Black carbon concentrations when travelling by car were 1.4 to 2.0 times higher than in the other transport modes due to the closer proximity to exhaust emissions. There are marked differences in PM chemical composition depending on transport mode. In particular, Fe was the most abundant component of metro PM, derived from abrasion of rail-wheel-brake interfaces. Enhanced concentrations of Zn and Cu in cars and buses were related with brake and tyre wear particles, which can penetrate into the vehicles. In the motorised transport modes, Fe, Zn, Cu, Ni and K were correlated, evidencing their common traffic-related source. On average, the highest inhaled dose of PM2.5 was observed while cycling (55 µg), and the lowest in car travels (17 µg). Cyclists inhaled higher doses of PM2.5 due to both higher inhalation rates and longer journey times, with a clear enrichment in mineral elements. The presented results evidence the importance of considering the transport mode in exposure assessment studies.

Relationship between indoor and outdoor size-fractionated particulate matter in urban microenvironments: Levels, chemical composition and sources.

Exposure to particulate matter (PM) has been associated with adverse health outcomes, particularly in susceptible population groups such as children. This study aims to characterise children's exposure to PM and its chemical constituents. Size-segregated aerosol samples (PM0.25, PM0.25-0.5, PM0.5-1.0, PM1.0-2.5 and PM2.5-10) were collected in the indoor and outdoor of homes and schools located in Lisbon (Portugal). Organic and elemental carbon (OC and EC) were determined by a thermo-optical method, whereas major and trace elements were analysed by X-Ray Fluorescence. In school, the children were exposed to higher PM concentrations than in home, which might be associated not only to the elevated human occupancy but also to outdoor infiltration. The pattern of PM mass size distribution was dependent on the location (home vs. school and indoor vs. outdoor). The presence of EC in PM0.25 and OC in PM0.25-0.5 was linked to traffic exhaust emissions. OC and EC in PM2.5-10 may be explained by their adhesion to the surface of coarser particles. Generally, the concentrations of mineral and marine elements increased with increasing PM size, while for anthropogenic elements happened the opposite. In schools, the concentrations of mineral matter, anthropogenic elements and marine aerosol were higher than in homes. High mineral matter concentrations found in schools were related to the close proximity to busy roads and elevated human occupancy. Overall, the results suggest that exposure to PM is relevant and highlights the need for strategies that provide healthier indoor environments, principally in schools.

Filters from taxis air conditioning system: A tool to characterize driver's occupational exposure to bioburden?

Bioburden proliferation in filters from air conditioning systems of taxis represents a possible source of occupational exposure. The aim of this study was to determine the occurrence of fungi and bacteria in filters from the air conditioning system of taxis used for patient transportation and to assess the exposure of drivers to bioburden. Filters from the air conditioning systems of 19 taxis and 28 personal vehicles (used as controls) operating in three Portuguese cities including the capital Lisbon, were collected during the winter season. The occurrence and significance of bioburden detected in the different vehicles are reported and discussed in terms of colony-forming units (CFU) per 1 m2 of filter area and by the identification of the most frequently detected fungal isolates based on morphology. Azole-resistant mycobiota, fungal biomass, and molecular detection of Aspergillus species/strains were also determined. Bacterial growth was more prevalent in taxis (63.2%) than in personal vehicles (26.3%), whereas fungal growth was more prevalent in personal vehicles (53.6%) than in taxis (21.1-31.6%). Seven different azole-resistant species were identified in this study in 42.1% taxi filters. Levels of fungal biomass were above the detection limit in 63% taxi filters and in 75% personal vehicle filters. No toxigenic species were detected by molecular analysis in the assessed filters. The results obtained show that bioburden proliferation occurs widely in filters from the air conditioning systems of taxis, including the proliferation of azole-resistant fungal species, suggesting that filters should be replaced more frequently. The use of culture based-methods and molecular tools combined enabled an improved risk characterization in this setting.

Fungal burden exposure assessment in podiatry clinics from Ireland.

Fungi are amongst the bioaerosols of most importance, as indicated by the growing interest in this field of research. The aim was to characterize the exposure to fungal burden in podiatry clinics using culture-based and molecular methods. METHODS: Airborne fungi were collected using an impaction air sampler and surface samples were also performed. Fourteen air samples were collected for direct detection of fungal DNA from filamentous fungi and dermatophytes. Overall, 63.6 % of the evening samples and 46 % of the morning samples surpassed the threshold values (150 CFU/m3). Molecular detection, by real time PCR, of the target fungal species/strains (Aspergillus and Stachybotrys species) was negative for all samples collected. Trichophyton rubrum was detected by PCR analysis in one DNA sample collected on day six. Results suggest the use of both culture-based and molecular methodologies are desirable for a complete evaluation of fungal burden in this particular health care setting.

Fungal contamination in green coffee beans samples: A public health concern.

Studies on the microbiology of coffee cherries and beans have shown that the predominant toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants. The aim of this study was to investigate the distribution of fungi in Coffea arabica L. (Arabica coffee) and Coffea canephora L. var. robusta (Robusta coffee) green coffee samples obtained from different sources at the pre-roasting stage. Twenty-eight green coffee samples from different countries of origin (Brazil, Timor, Honduras, Angola, Vietnam, Costa Rica, Colombia, Guatemala, Nicaragua, India, and Uganda) were evaluated. The fungal load in the contaminated samples ranged from 0 to 12330 colony forming units (CFU)/g, of which approximately 67% presented contamination levels below 1500 CFU/g, while 11% exhibited intermediate contamination levels between 1500 and 3000 CFU/g. Contamination levels higher than 3000 CFU/g were found in 22% of contaminated coffee samples. Fifteen different fungi were isolated by culture-based methods and Aspergillus species belonging to different sections (complexes). The predominant Aspergillus section detected was Nigri (39%), followed by Aspergillus section Circumdati (29%). Molecular analysis detected the presence of Aspergillus sections Fumigati and Circumdati. The% coffee samples where Aspergillus species were identified by culture-based methods were 96%. Data demonstrated that green coffee beans samples were contaminated with toxigenic fungal species. Since mycotoxins may be resistant to the roasting process, this suggests possible exposure to mycotoxins through consumption of coffee. Further studies need to be conducted to provide information on critical points of coffee processing, such that fungal contamination may be reduced or eliminated and thus exposure to fungi and mycotoxins through coffee handling and consumption be prevented.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

Aspergillus spp. prevalence in different Portuguese occupational environments: What is the real scenario in high load settings?

The genus Aspergillus is one of the most prevalent regarding fungi in several highly contaminated occupational environments. The goal of the current study was to assess the prevalence of Aspergillus spp. in different settings, focusing on those where a higher load of fungal contamination is expected according to the European Agency for Safety and Health at Work. A specific protocol to ensure a more accurate assessment of the exposure to Aspergillus spp. is proposed aimed at allowing a detailed risk characterization and management. Two wastewater treatment plants, one wastewater elevation plant, four waste treatment plants, three cork industries, five slaughter houses, four feed industries, one poultry pavilion, and two swineries, all located in the outskirts of Lisbon, were assessed. In total, 125 air samples and 125 surface samples were collected and analysed by culture-based methods. Real-time polymerase chain reaction was performed to detect fungal presence in 100 samples, targeting the Aspergillus sections Circumdati, Flavi, and Fumigati. The highest prevalence of Aspergillus spp. was found in wastewater treatment plants (69.3%; 31.1%), waste treatment plants (34.8%; 73.6%), and poultry feed industry (6.3%; 26.1%), in air and surfaces, respectively. Aspergillus spp. was also prevalent in cork industry (0.9%; 23.4%), slaughter houses (1.6%; 17.7%), and swineries (7.4%; 9.5%), in air and surfaces, respectively. The Aspergillus sections more prevalent in the air and surfaces of all the assessed settings were the Nigri section (47.46%; 44.71%, respectively), followed by Fumigati (22.28%; 27.97%, respectively) and Flavi (10.78%; 11.45%, respectively) sections. Aspergillus section Fumigati was successfully amplified by qPCR in 18 sampling sites where the presence of this fungal species had not been identified by conventional methods. It should be highlighted that the occupational exposure burden is due not only to the Aspergillus load, but also to the toxigenic potential of this genus. Based on our results, a protocol relied in the application of conventional and molecular methods in parallel is herein suggested aimed at allowing a better risk characterization and management.

A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation.

Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer's and Parkinson's. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo.

Occupational exposure to fungi and particles in animal feed industry.

BACKGROUND: Very few studies regarding fungal and particulate matter (PM) exposure in feed industry have been reported, although such contaminants are likely to be a significant contributing factor to several symptoms reported among workers. The purpose of this study has been to characterize fungal and dust exposure in one Portuguese feed industry. MATERIAL AND METHODS: Air and surface samples were collected and subject to further macro- and microscopic observations. In addition we collected other air samples in order to perform real-time quantitative polymerase chain reaction (PCR) amplification of genes from Aspergillus fumigatus and Aspergillus flavus complexes as well as Stachybotrys chartarum. Additionally, two exposure metrics were considered - particle mass concentration (PMC), measured in 5 different sizes (PM0.5, PM1, PM2.5, PM5, PM10), and particle number concentration (PNC) based on results given in 6 different sizes in terms of diameter (0.3 µm, 0.5 µm, 1 µm, 2.5 µm, 5 µm and 10 µm). RESULTS: Species from the Aspergillus fumigatus complex were the most abundant in air (46.6%) and in surfaces, Penicillium genus was the most frequently found (32%). The only DNA was detected from A. fumigatus complex. The most prevalent in dust samples were smaller particles which may reach deep into the respiratory system and trigger not only local effects but also the systemic ones. CONCLUSIONS: Future research work must be developed aiming at assessing the real health effects of these co-exposures. Med Pr 2016;67(2):143-154.

The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model.

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's or Parkinson's disease. Transthyretin (TTR) is a homotetrameric protein implicated in several amyloidoses like Senile Systemic Amyloidosis (SSA), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), and the rare Central Nervous System selective Amyloidosis (CNSA). In this work, we have investigated the kinetics of TTR aggregation into amyloid fibrils produced by the addition of NaCl to acid-unfolded TTR monomers and we propose a mathematically simple kinetic mechanism to analyse the aggregation kinetics of TTR. We have conducted circular dichroism, intrinsic tryptophan fluorescence and thioflavin-T emission experiments to follow the conformational changes accompanying amyloid formation at different TTR concentrations. Kinetic traces were adjusted to a two-step model with the first step being second-order and the second being unimolecular. The molecular species present in the pathway of TTR oligomerization were characterized by size exclusion chromatography coupled to multi-angle light scattering and by transmission electron microscopy. The results show the transient accumulation of oligomers composed of 6 to 10 monomers in agreement with reports suggesting that these oligomers may be the causative agent of cell toxicity. The results obtained may prove to be useful in understanding the mode of action of different compounds in preventing fibril formation and, therefore, in designing new drugs against TTR amyloidosis.

Fungal burden in waste industry: an occupational risk to be solved.

High loads of fungi have been reported in different types of waste management plants. This study intends to assess fungal contamination in one waste-sorting plant before and after cleaning procedures in order to analyze their effectiveness. Air samples of 50 L were collected through an impaction method, while surface samples, taken at the same time, were collected by the swabbing method and subject to further macro- and microscopic observations. In addition, we collected air samples of 250 L using the impinger Coriolis µ air sampler (Bertin Technologies) at 300 L/min airflow rate in order to perform real-time quantitative PCR (qPCR) amplification of genes from specific fungal species, namely Aspergillus fumigatus and Aspergillus flavus complexes, as well as Stachybotrys chartarum species. Fungal quantification in the air ranged from 180 to 5,280 CFU m(-3) before cleaning and from 220 to 2,460 CFU m(-3) after cleaning procedures. Surfaces presented results that ranged from 29×10(4) to 109×10(4) CFU m(-2) before cleaning and from 11×10(4) to 89×10(4) CFU m(-2) after cleaning. Statistically significant differences regarding fungal load were not detected between before and after cleaning procedures. Toxigenic strains from A. flavus complex and S. chartarum were not detected by qPCR. Conversely, the A. fumigatus species was successfully detected by qPCR and interestingly it was amplified in two samples where no detection by conventional methods was observed. Overall, these results reveal the inefficacy of the cleaning procedures and that it is important to determine fungal burden in order to carry out risk assessment.

Portuguese cork industry: filling the knowledge gap regarding occupational exposure to fungi and related health effects.

Introduction: The presence of the Penicillium section Aspergilloides (formerly known as Penicillium glabrum) in the cork industry involves the risk of respiratory diseases such as suberosis. Methods: The aim of this study was to corroborate the predominant fungi present in this occupational environment by performing a mycological analysis of 360 workers' nasal exudates collected by nasal swabs. Additionally, evaluation of respiratory disorders among the cork workers was also performed by spirometry. Results: Penicillium section Aspergilloides was detected by qPCR in 37 out of the 360 nasal swabs collected from workers' samples. From those, 25 remained negative for Penicillium sp. when using culture-based methods. A significant association was found between ventilatory defects and years of work in the cork industry, with those people working for 10 or more years in this industry having an approximately two-fold increased risk of having ventilatory defects compared to those working less time in this setting. Among the workers who detected the presence of Penicillium section Aspergilloides, those with symptoms presented slightly higher average values of CFU. Discussion: Overall, the results obtained in this study show that working in the cork industry may have adverse effects on worker's respiratory health. Nevertheless, more studies are needed (e.g., using serological assays) to clarify the impact of each risk factor (fungi and dust) on disease etiology.

Source apportionment of children daily exposure to particulate matter.

The present study aims to investigate the sources of particulate pollution in indoor and outdoor environments, with focus on determining their contribution to the exposure of children to airborne particulate matter (PM). To this end, parallel indoor and outdoor measurements were carried out for a selection of 40 homes and 5 schools between September 2017 and October 2018. PM2.5 and PM2.5-10 samples were collected during five days in each microenvironment (ME) and analysed by X-Ray Fluorescence (XRF), for the determination of elements, and by a thermal-optical technique, for the measurement of organic and elemental carbon. The source apportionment analysis of the PM composition data, by means of the receptor model SoFi (Source Finder) 8 Pro, resulted in the identification of nine sources: exhaust and non-exhaust emissions from traffic, secondary particles, heavy oil combustion, industry, sea salt, soil, city dust, and an indoor source characterized by high levels of organic carbon. Integrated daily exposure to PM2.5 was on average 21 µg/m3. The organic matter, resulting from cleaning, cooking, smoking and biological material, was the major source contributing by 31% to the PM2.5 exposure. The source city dust, which was highly influenced by the resuspension of dust in classrooms, was the second main source (26%), followed by traffic (24%). The major sources affecting the integrated exposure to PM10, which was on average 33 µg/m3, were the city dust (39%), indoor organics (24%) and traffic (16%). This study provides important information for the design of measures to reduce the exposure of children to PM.

Assessing Multi-Attribute Characterization of Enveloped and Non-Enveloped Viral Particles by Capillary Electrophoresis.

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.

Advances in Lentivirus Purification.

Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.

Bioburden in sleeping environments from Portuguese dwellings.

A wider characterization of indoor air quality during sleep is still lacking in the literature. This study intends to assess bioburden before and after sleeping periods in Portuguese dwellings through active methods (air sampling) coupled with passive methods, such as electrostatic dust cloths (EDC); and investigate associations between before and after sleeping and bioburden. In addition, and driven by the lack of information regarding fungi azole-resistance in Portuguese dwellings, a screening with supplemented media was also performed. The most prevalent genera of airborne bacteria identified in the indoor air of the bedrooms were Micrococcus (41%), Staphylococcus (15%) and Neisseria (9%). The major indoor bacterial species isolated in all ten studied bedrooms were Micrococcus luteus (30%), Staphylococcus aureus (13%) and Micrococcus varians (11%). Our results highlight that our bodies are the source of the majority of the bacteria found in the indoor air of our homes. Regarding air fungal contamination, Chrysosporium spp. presented the highest prevalence both in after the sleeping period (40.8%) and before the sleeping period (28.8%) followed by Penicillium spp. (23.47% morning; 23.6% night) and Chrysonilia spp. (12.4% morning; 20.3% night). Several Aspergillus sections were identified in air and EDC samples. However, none of the fungal species/strains (Aspergillus sections Fumigati, Flavi, Nidulantes and Circumdati) were amplified by qPCR in the analyzed EDC. The correlations observed suggest reduced susceptibility to antifungal drugs of some fungal species found in sleeping environments. Toxigenic fungal species and indicators of harmful fungal contamination were observed in sleeping environments.

Analysis of spatial factors, time-activity and infiltration on outdoor generated PM2.5 exposures of school children in five European cities.

Atmospheric particles are a major environmental health risk. Assessments of air pollution related health burden are often based on outdoor concentrations estimated at residential locations, ignoring spatial mobility, time-activity patterns, and indoor exposures. The aim of this work is to quantify impacts of these factors on outdoor-originated fine particle exposures of school children. We apply nested WRF-CAMx modelling of PM2.5 concentrations, gridded population, and school location data. Infiltration and enrichment factors were collected and applied to Athens, Kuopio, Lisbon, Porto, and Treviso. Exposures of school children were calculated for residential and school outdoor and indoor, other indoor, and traffic microenvironments. Combined with time-activity patterns six exposure models were created. Model complexity was increased incrementally starting from residential and school outdoor exposures. Even though levels in traffic and outdoors were considerably higher, 80-84% of the exposure to outdoor particles occurred in indoor environments. The simplest and also commonly used approach of using residential outdoor concentrations as population exposure descriptor (model 1), led on average to 26% higher estimates (15.7 µg/m3) compared with the most complex model (# 6) including home and school outdoor and indoor, other indoor and traffic microenvironments (12.5 µg/m3). These results emphasize the importance of including spatial mobility, time-activity and infiltration to reduce bias in exposure estimates.