Human Papillomavirus Minor Capsid Protein L2 Mediates Intracellular Trafficking into and Passage beyond the Endoplasmic Reticulum.

Human papillomaviruses (HPVs) consist of two capsid proteins: major capsid protein L1 and minor capsid protein L2. The L2 protein has been shown to be involved in intracellular trafficking events that lead to the deposition of the viral DNA into the nucleus. In this study, we investigate the role of HPV16 L2 residues 43-DQILQ-47 during intracellular trafficking in human keratinocytes. We demonstrate that the highly conserved amino acids aspartic acid, isoleucine, and leucine are involved with the intracellular trafficking of the virus. Amino acid substitution of the isoleucine and leucine residues with alanine residues results in a significant decrease in infectivity of the pseudovirions without any changes to the binding or internalization of the virus. The pseudovirions containing these substitutions exhibit an altered trafficking pattern and do not deposit the viral pseudogenome into the nucleus. Instead, these mutated pseudovirions display a lack of interaction with syntaxin 18, an ER SNARE protein, are unable to progress past the endoplasmic reticulum (ER) and are redirected to the lysosomes. The results of this study help to elucidate the role and potential involvement of the 43-DQILQ-47 sequence during intracellular trafficking, specifically during trafficking beyond the ER. IMPORTANCE High-risk types of human papillomaviruses (HPVs), such as HPV16, are highly associated with cervical, anogenital, and oropharyngeal cancers. The minor capsid protein L2 is essential for the intracellular trafficking of the viral DNA to the nucleus. This study investigates the role of amino acid residues 43-DQILQ-47 of the HPV16 L2 protein in the intracellular trafficking of the virus. Understanding how the virus traffics through the cell is a key factor in the development of additional preventative antiviral therapies. This study illustrates, through modification of the 43-DQILQ-47 sequence in pseudovirions, the importance of the 43-DQILQ-47 sequence in the trafficking of the virus beyond the endoplasmic reticulum.

One-year follow-up of microbial diversity in bioaerosols emitted in a waste sorting plant in France.

Bioaerosols emitted in waste sorting plants (WSP) can induce some adverse health effects on the workers such as rhinitis, asthma and hypersensitivity pneumonitis. The composition of these bioaerosols is scarcely known and most of the time assessed using culture-dependent methods. Due to the well-known limitations of cultural methods, these biodiversity measurements underestimate the actual microbial taxon richness. The aim of the study was to assess the airborne microbial biodiversity by using a sequencing method in a French waste sorting plant (WSP) for one year and to investigate the main factors of variability of this biodiversity. Static sampling was performed in five areas in the plant and compared to an indoor reference (IR), using closed-face cassettes (10 L.min-1) with polycarbonate membranes, every month for one year. Environmental data was measured (temperature, relative humidity). After DNA extraction, microbial biodiversity was assessed by means of sequencing. Bacterial genera Staphylococcus, Streptococcus, Prevotella, Lactococcus, Lactobacillus, Pseudomonas and fungal genera Wallemia, Cladosporium, Debaryomyces, Penicillium, Alternaria were the most predominant airborne microorganisms. Microbial biodiversity was different in the plant compared to the IR and seemed to be influenced by the season.

Transportation can cancel out the ecological advantages of producing organic cacao: The carbon footprint of the globalized agrifood system of ecuadorian chocolate.

Under the hypothesis that organically managed cacao agroforestry systems report a lower global warming potential (GWP) and reduce other environmental pressure indicators compared with conventionally managed systems and monocultures, this work discusses how global transportation can cut back the ecological advantage of the production phase. For this purpose, the life cycle assessment (LCA) of 1 kg of dark chocolate manufactured with Ecuadorian cacao has been performed (cradle-to-retailer approach), including the indirect impacts of transportation and estimating the equilibrium distances beyond which organic chocolate would have a higher impact than chocolate manufactured from cacao grown in monocultures and/or conventionally managed systems. To articulate the discussion, the carbon footprint (CF) of cacao/chocolate was analyzed together with 10 additional LCA-related impact categories. Three management systems-conventional monoculture (CM) and agroforestry (CA), and organic agroforestry (OA)-and three different supply chain scenarios with different weights in the transportation phase were studied. Expanding on the concept of "food miles", the equivalent kilometers of the impact of emissions (km-eq) (or cumulated energy demand, eutrophication, etc.) were defined as the variable distance that a certain means of transportation can travel in relation to a fixed level of GHG emissions (or MJ, kg PO4-eq, etc.). The CF of the life cycle of cacao/chocolate was estimated at between 2.04 and 4.66 kg CO2-eq kg-1. The relative weight of transportation in relation to the total GHG emissions ranged between 8.9% and 51.1%, with cacao/chocolate traveling between 1380 and 9155 km-eq. The CF of chocolate made from cacao grown in OA systems was 22.7%-34.2% and 6.3%-10.7% lower than the CF of chocolate produced from cacao grown in CM and CA and manufactured and transported under the same conditions. The equilibrium distances between managements were estimated at 1213 and 5275 km-eq. Beyond those equivalent kilometers, organic chocolate would have a larger CF than chocolate manufactured from cacao grown, respectively, in CA and CM systems. Our results indicate that transportation would cancel out this and most other comparative ecological advantages of producing organic cacao analyzed in this work. Directly exporting chocolate from cacao-producing countries and relocating chocolate manufacture would help reduce GHG emissions and other environmental impacts of the supply chain.

Effects of plant features on symptoms and airway inflammation in compost workers followed over 18 months.

This study investigated the plant features associated with increased irritation symptoms and levels of inflammation markers among compost workers (CWs). Ninety CWs were followed over 18 months, using questionnaires on respiratory symptoms, fractional exhaled nitric oxide measurements, spirometry, a methacholine bronchial challenge test, and quantification of specific immunoglobulins E (IgE) and G. CWs in plants processing the highest quantities of waste exhibited more airway irritation symptoms. So did the CWs in partially and fully indoor plants as compared to those in plants entirely outdoors. Working in sewage sludge versus green waste plants and having a high level of exposure were associated with higher levels of different IgE. The duration of employment decreased the FEV1 by 16 ml per year. Working in an indoor plant is linked to symptoms and inflammation markers in CWs.

First Metagenomic Survey of the Microbial Diversity in Bioaerosols Emitted in Waste Sorting Plants.

Waste sorting activities are source of occupational bioaerosol exposures that are associated with several health disorders. New analytical tools, based on next-generation sequencing (NGS) technologies, provide powerful methods to assess the microbial composition of bioaerosols. The objectives of the study were (i) to assess the feasibility and the repeatability of NGS-based biodiversity measurements and (ii) to study the microbial biodiversity using NGS in bioaerosols emitted in a waste sorting plant (WSP). Three stationary parallel samples were collected in a sorting cabin using closed-face cassettes equipped with polycarbonate membranes. Bacterial and fungal diversity was assessed by sequencing 16S and 18S rDNA genes using either Illumina sequencing or 454 pyrosequencing methods. At sampling point, airborne bacteria were dominated by Proteobacteria, Firmicutes, and Actinobacteria with prevailing genera assigned to unclassified Enterobacteriaceae, Staphylococcus, Acinetobacter, Leuconostoc, Pseudomonas, and Lactobacillus. Airborne fungi were dominated by Ascomycota with prevailing genera assigned to Penicillium, Aspergillus, Rhizopus, Wallemia, and Hemicarpenteles. The NGS biodiversity measurements revealed a higher biodiversity bioaerosols that previously reported for WSP in studies carried out using culture methods followed by identification of microorganisms. These results provide the first survey about taxonomic biodiversity in bioaerosols from WSPs using high-throughput sequencing.

Sampling Efficiency and Performance of Selected Thoracic Aerosol Samplers.

Measurement of worker exposure to a thoracic health-related aerosol fraction is necessary in a number of occupational situations. This is the case of workplaces with atmospheres polluted by fibrous particles, such as cotton dust or asbestos, and by particles inducing irritation or bronchoconstriction such as acid mists or flour dust. Three personal and two static thoracic aerosol samplers were tested under laboratory conditions. Sampling efficiency with respect to particle aerodynamic diameter was measured in a horizontal low wind tunnel and in a vertical calm air chamber. Sampling performance was evaluated against conventional thoracic penetration. Three of the tested samplers performed well, when sampling the thoracic aerosol at nominal flow rate and two others performed well at optimized flow rate. The limit of flow rate optimization was found when using cyclone samplers.

Endotoxin deposits on the inner surfaces of closed-face cassettes during bioaerosol sampling: a field investigation at composting facilities.

A set of 270 bioaerosol samples was taken from 15 composting facilities using polystyrene closed-face filter cassettes (CFCs). The objective was to measure the quantity of endotoxin deposits on the inner surfaces of the cassettes (sometimes referred to as 'wall deposits'). The results show that endotoxins are deposited on the inner surfaces of the CFCs through sampling and/or handling of samples. The quantity of endotoxins measured on inner surfaces range between 0.05 (the limit of detection of the method) and 3100 endotoxin units per cassette. The deposits can represent a large and variable percentage of the endotoxins sampled. More than a third of the samples presented a percentage of inner surface deposits >40% of the total quantity of endotoxins collected (filter + inner surfaces). Omitting these inner surface deposits in the analytical process lead to measurement errors relative to sampling all particles entering the CFC sampler, corresponding to a developing consensus on matching the inhalable particulate sampling convention. The result would be underestimated exposures and could affect the decision as to whether or not a result is acceptable in comparison to airborne concentration limits defined in terms of the inhalability convention. The results of this study suggest including the endotoxins deposited on the inner surfaces of CFCs during analysis. Further researches are necessary to investigate endotoxin deposits on the inner cassette surfaces in other working sectors.

Assessment of workers' exposure to bioaerosols in a French cheese factory.

Hundreds of different cheeses are produced in France, where 23.9kg of cheese were consumed per inhabitant in 2009, when it was ranked the second cheese-consuming nation. To meet this considerable demand, a large number of cheese factories exist where many workers, especially cheese washers, may be exposed to fungal bioaerosols that can lead to adverse toxinic and allergic effects. Airborne bacteria, fragments, or microbial by-products (endotoxins) are also found and contribute to total worker exposure. However, there is almost no published data concerning worker exposure or characteristics of bioaerosols emitted during these activities. Here, we measured the parameters (concentrations, species present, and size distribution) of the culturable fungal bioaerosol emitted in a French natural-rind cheese-maturing cellar. Concentrations of airborne bacteria and endotoxins were also measured. The main tasks were investigated using stationary or personal sampling over three consecutive days. Depending on the work area, high concentrations of culturable mesophilic microorganisms were measured (using closed-face cassettes): from 10(4) to 2×10(8) CFU m(-3) for fungi and from 10(3) to 10(6) CFU m(-3) for bacteria. These concentrations are 10- to 100000-fold higher than those measured at two reference points (indoor and outdoor) that are assumed not to be contaminated by the plant's activities. Endotoxin concentrations were between 10 and 300 EU m(-3) in the plant. Exposure was further assessed by identifying the predominant culturable fungi (allergenic Mucor fuscus and Penicillium sp.) and by measuring particle size distributions (cascade impactor). Airborne fungal entities (spores, mycelium strands and fragments, agglomerates, etc.) were found with aerodynamic diameters from 3 to over 20 µm. A metrological approach was used to fully characterize the culturable fungal aerosols generated during cheese maturing in this plant. The results show that workers are exposed to concentrations of airborne culturable fungi, sometimes very high, throughout the manufacturing process. In addition to fungi, culturable bacteria and endotoxins are also present in the work atmosphere. All these microbial organisms thus contribute in a complex manner to total worker exposure. Despite the lack of both occupational exposure limit values and standardized measuring methods, our results suggest that an immunological risk may occur among workers, especially for cheese brushers, cheese washers, and packagers who are the most exposed workers in the factory.

Development and evaluation of a method for the quantification of airborne Thermoactinomyces vulgaris by real-time PCR.

Actinomycetes are ubiquitous and some can be potentially pathogenic for humans when present in the air of some working areas. It's notably the case for Thermoactinomyces vulgaris in composting facilities where aerial concentrations can reach high values of more than 10(7) CFU·m(-3). Workers exposure to these inhalable bioaerosols can be the source of various diseases. The literature reveals a lack of knowledge about risk assessment: there is neither dose-effects relationship for most agents, or threshold limit value. The objectives of this study were to develop and standardize a method to quantify workers exposure to bioaerosols. We have developed and evaluated a method to quantify airborne T. vulgaris based on DNA extraction of aerial microbial communities and qPCR. Four DNA extraction protocols were compared, and primers and a hydrolysis probe were designed for specific amplification of the target species (gyrB gene). This method was compared to traditional methods based on viable or cultivable counting by epifluorescence microscopy or plating on selective media. The method was applied on environmental bioaerosols sampled under real exposure conditions in composting plants. We demonstrate that the method to quantify T. vulgaris in bioaerosols is specific, sensitive and repeatable. We demonstrate the occurrence and quantified T. vulgaris in the atmosphere of composting facilities with concentrations ranging from 3×10(2) to 3×10(6)×m(-3).

Documentation of bioaerosol concentrations in an indoor composting facility in France.

Bioaerosol concentrations were investigated in a totally indoor composting facility processing fermentable household and green wastes to assess their variability. Stationary samples were collected by filtration close to specific composting operations and then were analysed for cultivable mesophilic bacteria, thermophilic bacteria, mesophilic fungi, thermophilic fungi, endotoxins and total airborne bacteria (DAPI-staining). Indoor concentrations exceeded the background levels, between 500 and 5400 EU m(-3) for endotoxins, 10(4) and 10(6) CFU m(-3) for cultivable bacteria and generally below 10(5) CFU m(-3) for airborne cultivable fungi. No significant (p > 0.05) differences were observed between the indoor composting operations. Successive 30 minute bioaerosol samples were collected to investigate the variation of cultivable mesophilic microorganisms over the work shift. Concentrations of mesophilic bacteria and fungi varied up to 1 log unit depending on the time at which they were collected in the day. Total airborne particles, counted using an optical particle counter, were present at up to 10(8) particles m(-3) and several concentration peaks were noted. Values for total airborne bacteria were roughly 70-fold higher than cultivable bacteria. These results raise the question of the sampling strategy (duration of sampling; number of samples to be collected) used in similar studies. They provide new bioaerosol concentration data in a composting facility and suggest that the filtration sampling method might be a useful tool for exposure measurements in that occupational environment.

Workplace aerosol mass concentration measurement using optical particle counters.

Direct-reading aerosol measurement usually uses the optical properties of airborne particles to detect and measure particle concentration. In the case of occupational hygiene, mass concentration measurement is often required. Two aerosol monitoring methods are based on the principle of light scattering: optical particle counting (OPC) and photometry. The former analyses the light scattered by a single particle, the latter by a cloud of particles. Both methods need calibration to transform the quantity of scattered light detected into particle concentration. Photometers are simpler to use and can be directly calibrated to measure mass concentration. However, their response varies not only with aerosol concentration but also with particle size distribution, which frequently contributes to biased measurement. Optical particle counters directly measure the particle number concentration and particle size that allows assessment of the particle mass provided the particles are spherical and of known density. An integrating algorithm is used to calculate the mass concentration of any conventional health-related aerosol fraction. The concentrations calculated thus have been compared with simultaneous measurements by conventional gravimetric sampling to check the possibility of field OPC calibration with real workplace aerosols with a view to further monitoring particle mass concentration. Aerosol concentrations were measured in the food industry using the OPC GRIMM® 1.108 and the CIP 10-Inhalable and CIP 10-Respirable (ARELCO®) aerosol samplers while meat sausages were being brushed and coated with calcium carbonate. Previously, the original OPC inlet had been adapted to sample inhalable aerosol. A mixed aerosol of calcium carbonate and fungi spores was present in the workplace. The OPC particle-size distribution and an estimated average particle density of both aerosol components were used to calculate the mass concentration. The inhalable and respirable aerosol fractions calculated from the OPC data are closely correlated with the results of the particle size-selective sampling using the CIP 10. Furthermore, the OPC data allow calculation of the thoracic fraction of workplace aerosol (not measured by sampling), which is interesting in the presence of allergenic particles like fungi spores. The results also show that the modified COP inlet adequately samples inhalable aerosol in the range of workplace particle-size distribution.

Laboratory study of selected personal inhalable aerosol samplers.

Assessment of inhalable dust exposure requires reliable sampling methods in order to measure airborne inhalable particles' concentrations. Many inhalable aerosol samplers can be used but their performances widely vary and remain unknown in some cases. The sampling performance of inhalable samplers is strongly dependent on particle size and ambient air velocity. Five inhalable aerosol samplers have been studied in two laboratory wind tunnels using polydisperse glass-beads' test aerosol. Samplers tested were IOM sampler (UK), two versions of CIP 10-I sampler, v1 and v2 (F), 37-mm closed face cassette sampler (USA), 37-mm cassette fitted up with an ACCU-CAP insert (USA), and Button sampler (USA). Particle size-dependent sampling efficiencies were measured in a horizontal wind tunnel under a 1 m s(-1) wind velocity and in a vertical tunnel under calm air, using a specific method with Coulter(R) counter particle size number distribution determinations. Compared with CEN-ISO-ACGIH sampling criteria for inhalable dust, the experimental results show fairly high sampling efficiency for the IOM and CIP 10-I v2 samplers and slightly lower efficiencies for the Button and CIP 10-I v1 samplers. The closed face cassette (4-mm orifice) produced the poorest performances of all the tested samplers. This can be improved by using the ACCU-CAP internal capsule, which prevents inner wall losses inside the cassette. Significant differences between moving air and calm air sampling efficiency were observed for all the studied samplers.

Site comparison of selected aerosol samplers in the wood industry.

Several samplers (IOM, CIP 10-I v1, ACCU-CAP, and Button) were evaluated at various wood industry companies using the CALTOOL system. The results obtained show that compared to the CALTOOL mouth, which can be considered to be representative of the exposure of a person placed at the same location under the same experimental conditions, the concentrations measured by the IOM, CIP 10-I v1, and ACCU-CAP samplers are not significantly different (respectively, 1.12, 0.94, and 0.80 compared to 1.00), the Button sampler (0.86) being close to the ACCU-CAP sampler. Comparisons of dust concentrations measured using both a closed-face cassette (CFC) and one of the above samplers were also made. In all, 235 sampling pairs (sampler + CFC) taken at six companies provided us with a comparison of concentrations measured using IOM, CIP 10-I v1, ACCU-CAP, and Button samplers with concentrations measured using a CFC. All the studied samplers collected systematically more dust than the CFC (2.0 times more for the IOM sampler, 1.84 times more for the CIP 10-I v1 sampler, 1.68 times more for the ACCU-CAP sampler, and 1.46 times more for the Button sampler). The literature most frequently compares the IOM sampler with the CFC: published results generally show larger differences compared with the CFC than those found during our research. There are several explanations for this difference, one of which involves CFC orientation during sampling. It has been shown that concentrations measured using a CFC are dependent on its orientation. Different CFC positions from one sampling session to another are therefore likely to cause differences during CFC-IOM sampler comparisons.