A Metagenomic Framework for the Study of Airborne Microbial Communities

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.     

Wind-Mediated Spread of Low-Pathogenic Avian Influenza Virus into the Environment during Outbreaks at Commercial Poultry Farms

Avian influenza virus-infected poultry can release a large amount of virus-contaminated droppings that serve as sources of infection for susceptible birds. Much research so far has focused on virus spread within flocks. However, as fecal material or manure is a major constituent of airborne poultry dust, virus-contaminated particulate matter from infected flocks may be dispersed into the environment. We collected samples of suspended particulate matter, or the inhalable dust fraction, inside, upwind and downwind of buildings holding poultry infected with low-pathogenic avian influenza virus, and tested them for the presence of endotoxins and influenza virus to characterize the potential impact of airborne influenza virus transmission during outbreaks at commercial poultry farms. Influenza viruses were detected by RT-PCR in filter-rinse fluids collected up to 60 meters downwind from the barns, but virus isolation did not yield any isolates. Viral loads in the air samples were low and beyond the limit of RT-PCR quantification except for one in-barn measurement showing a virus concentration of 8.48x10⁴ genome copies/m³. Air samples taken outside poultry barns had endotoxin concentrations of ~50 EU/m³ that declined with increasing distance from the barn. Atmospheric dispersion modeling of particulate matter, using location-specific meteorological data for the sampling days, demonstrated a positive correlation between endotoxin measurements and modeled particulate matter concentrations, with an R² varying from 0.59 to 0.88. Our data suggest that areas at high risk for human or animal exposure to airborne influenza viruses can be modeled during an outbreak to allow directed interventions following targeted surveillance.     

Trace elements in the atmosphere.

The distribution and behaviour of particulate trace elements in the atmosphere have been studied by continuous measurements for 5 years at seven non-urban sites in the United Kingdom. Samples have been taken regularly of airborne dust, rainwater and dry deposition: these have been analysed for up to 36 elements. Concentrations of trace elements vary considerably between sites but the relative concentrations are among uniform: this suggests similarity of origin or good atmospheric mixing. By comparing the relative concentrations with those in soil it is possible to differentiate between trace elements that are derived from soil and those that may be attributed to industrial activity. This classification is supported by estimates of the particle sizes in air. The deposition of trace elements can be related to the concentrations presnet in soil and to the annual removal by crops. Retrospective analyses of stored samples from one site describe the history of trace element concentrations in air since 1957. The sea surface is considered as a possible source of atmospheric trace elements.     

Quantitative sampling of indoor air biomass by signature lipid biomarker analysis

Exposure to airborne biocontaminants may result in a multitude of health effects and is related to a pronounced increase in adult-onset asthma. Established culture-based procedures for quantifying microbial biomass from airborne environments have severe limitations. Assay of the phospholipid fatty acid (PLFA) components of airborne microorganisms provides a quantitative method to define biomass, community composition and nutritional/physiological activity of the microbial community. By collecting airborne particulate matter from a high volume via filtration, we collected sufficient biomass for quantitative PLFA analysis. Comparing high (filtration) and low (impaction) volume air sampling techniques at 26 locations within the Eastern United States, we determined that PLFA analysis provided a viable alternative to the established but flawed culture-based techniques for measuring airborne microbial biomass and community composition. Compared to the PLFA analysis, the culture techniques underestimated the actual viable airborne biomass present by between one to three orders of magnitude. A case study of a manufacturing plant at which there had been complaints regarding the indoor air quality is presented. Phospholipid fatty acid characterization of the biomass enabled contamination point source determination. In comparison with samples taken outdoors, increases in the relative proportion of trans PLFA, reflecting shifts in the physiological status of viable airborne Gram-negative bacteria, were detected in the indoor air samples at a majority of sampling sites. Received 29 September 1998/ Accepted in revised form 8 January 1999     

Blood meal induces global changes in midgut gene expression in the disease vector, Aedes aegypti

Blood feeding is an essential developmental process for many arthropods and plays a significant role in disease transmission. Understanding physiological responses in the midgut is important because it is the primary site of blood meal digestion and pathogenic infection. Processes that occur in the midgut in response to a blood meal have been studied but are poorly understood. Here, we use cDNA microarrays to examine midgut gene expression on a global level in response to blood feeding to assist in unraveling these processes. We have developed Aedes aegypti microarrays consisting of clones obtained from an expressed sequence tag project. Individual clones were amplified by polymerase chain reaction and printed onto glass slides. These microarrays were used to study the effects of a blood meal on midgut gene expression over a 72-h time course. As a result, a number of genes involved in processes such as nutrient uptake and metabolism, cellular stress responses, ion balance, and PM formation, as well as a number of unknown genes were induced or repressed in response to a blood meal based on this microarray data.     

The Efficient Method for Simultaneous Monitoring of the Culturable as Well as Nonculturable Airborne Microorganisms

Cultivation-based microbiological methods are a gold standard for monitoring of airborne micro-organisms to determine the occupational exposure levels or transmission paths of a particular infectious agent. Some highly contagious microorganisms are not easily culturable but it is becoming evident that cultivation and molecular methods are complementary and in these cases highly relevant. We report a simple and efficient method for sampling and analyzing airborne bacteria with an impactor-type high-flow-rate portable air sampler, currently used for monitoring culturable bacteria or fungi. A method is reported for extraction of nucleic acids from impacted cells without prior cultivation and using agarose as a sampling matrix. The DNA extraction efficiency was determined in spiked samples and, samples taken from a wastewater treatment plant and an alpine area. The abundance, diversity and quantity of total bacteria were analysed by a quantitative polymerase chain reaction, and by construction and analysis of clone libraries. The method does not interfere with downstream PCR analysis and can cover the gap between traditional culture and molecular techniques of bioaerosol monitoring.     

SlideToolkit: An Assistive Toolset for the Histological Quantification of Whole Slide Images

The demand for accurate and reproducible phenotyping of a disease trait increases with the rising number of biobanks and genome wide association studies. Detailed analysis of histology is a powerful way of phenotyping human tissues. Nonetheless, purely visual assessment of histological slides is time-consuming and liable to sampling variation and optical illusions and thereby observer variation, and external validation may be cumbersome. Therefore, within our own biobank, computerized quantification of digitized histological slides is often preferred as a more precise and reproducible, and sometimes more sensitive approach. Relatively few free toolkits are, however, available for fully digitized microscopic slides, usually known as whole slides images. In order to comply with this need, we developed the slideToolkit as a fast method to handle large quantities of low contrast whole slides images using advanced cell detecting algorithms. The slideToolkit has been developed for modern personal computers and high-performance clusters (HPCs) and is available as an open-source project on github.com. We here illustrate the power of slideToolkit by a repeated measurement of 303 digital slides containing CD3 stained (DAB) abdominal aortic aneurysm tissue from a tissue biobank. Our workflow consists of four consecutive steps. In the first step (acquisition), whole slide images are collected and converted to TIFF files. In the second step (preparation), files are organized. The third step (tiles), creates multiple manageable tiles to count. In the fourth step (analysis), tissue is analyzed and results are stored in a data set. Using this method, two consecutive measurements of 303 slides showed an intraclass correlation of 0.99. In conclusion, slideToolkit provides a free, powerful and versatile collection of tools for automated feature analysis of whole slide images to create reproducible and meaningful phenotypic data sets.     

Kinetic parameters in the compartmental analysis of lithium transport in Lemna gibba using the stable isotopes, 6Li and 7Li, as tracers

Due to the lack of a convenient radiotracer of Li, we have used the stable isotopes, ⁶Li and ⁷Li, for unidirectional flux measurements in Lemna gibba L. (GI). Detection was performed using an ionic microanalyser. The conventional method of compartmental analysis has been improved in several ways. Growth of the plant samples has been taken into consideration. The possible non-uniqueness of the kinetic solution fitting the experimental points has been considered by numerical calculation. The derivatives and not only the direct functions have been used in order to detect whether a constant term had to be added to the first-order terms. The validity of the estimates of the characteristic parameters of transport has been checked by using efflux and influx data simultaneously. The "manipulation-shock" effects have been shown not to be negligible. The kinetic data have no simple biological significance per se, but they can serve to calculate cellular parameters of transport when combined with appropriate stereometric data. These considerations are relevant not only to our present problem, but to any type of compartmental analysis, whatever the plant sample and the absorbed substrate.     

Health-related monitoring and assessment of airborne particulate matter

Since 1992, the International Atomic Energy Agency (IAEA) has been promoting studies of air pollution using a standard design of air sampler that provides separation on filters into two size fractions with cutoffs of 2.5 and 10 Μm (approximately). These are the size ranges presently considered to have the most important health consequences. Such filter samples are highly amenable to analysis using nuclear and related techniques. After reviewing some of the health effects of airborne particulate matter and current air quality standards and guidelines, this article provides an overview of current and recent IAEA programs in this area, which involve collaborative activities with participants in more than 40 countries.     

Semi-automated imaging system to quantitate Her-2/neu membrane receptor immunoreactivity in human breast cancer.

BACKGROUND: Immunocytochemical methods for quantitating Her-2/neu immunoreactivity rest on subjective semi-quantitative interpretations with resulting interobserver, intraobserver, and fatigue variability. METHODS: To standardize and quantitate measurements of Her-2/neu immunoreactivity, we created epithelial-recognition and specific membrane-recognition algorithms, which could image breast cancer cells against a background of stroma, compartmentalize the cancer cell into nucleus, cytoplasm and membrane, and quantitate the degree of Her-2/neu membrane immunoreactivity based on both gray scale intensity and RGB colorimetric determinations. Image acquisition utilized either scanner or microscope with attached camera with a resolution of 20 pixels/10 microm. Areas of 150 whole slides were screened and the regions of interest manually selected for image processing. Three hundred TMA cores were directly processed. Images were acquired by jpg conversion of svs virtual slides or direct jpg photomicrograph capture. Images were then assessed for quality and processed. RESULTS: The digital algorithms successfully created a semi-automated imaging system whose algorithm-based ordinal values for Her-2/neu both strongly correlated with the subjective measurements (intraclass correlation: 0.84; 95% confidence interval: 0.79-0.89) yet exhibited no run variability. In addition, the algorithms generated immunocytochemical measurements of Her-2/neu on an expanded continuous scale, which more reliably distinguished true Her-2/neu positivity from true Her-2/neu negativity (determined by FISH) than subjective or algorithmic ordinal scale measurements. Furthermore, the continuous scale measurements could better resolve different levels of Her-2/neu overexpression than either subjective or algorithmic ordinal interpretation. Other semi-automated analysis systems have been used to measure Her-2/neu and other cellular immunoreactivities, but these either have required proprietary hardware or have been based on luminosity differences alone. In contrast, our algorithms are independent of proprietary hardware and are based on not just luminosity but also many other imaging properties including epithelial recognition and membrane morphology. CONCLUSION: These features provide a more accurate, versatile, and robust imaging analysis platform.     

Measurement of resuspended aerosol in the Chernobyl area

Results of measurements of the resuspended radioactive aerosols in the Chernobyl area are presented which were obtained soon after the Chernobyl reactor accident and in a European project in 1992–1993. The measurements were carried out with the intention of obtaining a data base for dose assessment of resuspended radioactive particles. Potential significant dose contributions may result from inhalation and secondary contamination due to resuspended radionuclides. In this first article of a series of three papers, the instrumentation and the measurement uncertainties are discussed. An effort was made to sample quantitatively giant aerosol particles (particles larger than 10 μm aerodynamic diameter) as well. The comparison of the samplers shows, in general, an agreement of concentration measurements of ¹³⁷Cs and ⁷Be within a factor of two. One sampler was identified with larger discrepancies and needs additional investigation of its sampling characteristics; for another device, the recalibration of the analysing system is recommended. Ordinary integrating samplers have a loss of about 30% in ¹³⁷Cs activity compared to an isokinetic sampler collecting giant particles as well. The mean ratio of ¹³⁷Cs activity concentration between an instrument sampling only particles larger than 10 μm and an ordinary integrating sampler is 0.39 ± 0.15 during anthropogenic-enhanced resuspension. These findings demonstrate the significant contribution of giant particles to resuspended airborne radioactivity. The results of this study concerning integral measurements during wind-driven resuspension proved to be in good agreement with previously published data on resuspension. Received: 15 May 1997 / Accepted: 4 August 1997     

Components and results of a new preparation technique for automated analysis of cervical samples

Components and evaluations of a new preparative procedure for automated high-resolution analysis of cervical samples are presented. This procedure is based on sedimentation velocity separation of samples with subsequent fractionation of the separation column and centrifugal deposition of suspended cells on coated glass slides. A system for specimen collection and mailing of suspended samples is described. A new type of glass slide designed for automated analysis is presented, and centrifugal buckets for cell deposition on a 6-sq-cm area are described. Experimental results with different kinds of coating substances for glass slides as well as different isopyknic media are discussed, and data for differential cell counts are graphically demonstrated. Looking at the diagnostic accuracy and economic feasibility of this system, the authors realize that preparations have to be evaluated quantitatively and that constraints of sample size and processing time have to be taken into consideration for further developments.     

Nucleus image properties and cell death in MCF-10F cells grown on slide substrates differing in nature and size

Summary The immortalized human breast epithelial cell line MCF-10F is an important tool for studies on experimental tumorigenesis induced by drugs, transfected Ha-ras oncogene, and hormones. Considering that many relevant data have thus far been established only for MCF-10F cells cultivated on glass, and that there are data showing different cell death ratios for tumorigenic cells obtained from benzo[a]pyrene (BP)-transformed MCF-10F cells cultivated on plastic compared with glass, nuclear parameters estimated by image analysis and cell death ratios were compared for cells grown on plastic and glass substrates differing in chamber surface sizes and working culture medium volumes. It was concluded that for slides with a growth size equal to 9.4 cm², plastic substrate was more advantageous than glass for growing MCF-10F cells because although the apoptotic ratios (AR) for the cells grown on plastic are low as it would be expected for nontransformed cells, they are bigger than those reported for the BP-transformed MCF-10F cells cultivated on the same substrate but closer to those of the BP-transformed MCF-10F cells receiving a normal chromosome 17. In addition, the plastic substrate did not induce variable nuclear image results as those found in the latter. The 0.5-cm²-sized chambers on plastic slides proved to be inadequate for cell nuclear image analysis and cell death studies on account of the variable geometric, densitometric, and textural results and ARs produced and the unpublished consideration of a very slow growth rate generated under this growth condition.     

Computer-assisted sperm analysis of canine spermatozoa motility measurements

Computer-assisted sperm analysis (CASA) allows for the determination of specific motion characteristics of sperm cells in vitro. This study was designed to develop a system for the use of CASA to objectively evaluate canine sperm motility, and specifically to determine whether motility characteristics vary between individual dogs. Ejaculates from 10 dogs were collected weekly. Sperm cells were extended in a glucose-free TALP medium, placed on slides and videotaped at 200x. Videotaped samples were then analyzed by the Hamilton-Thorn Motility Analyzer, with 100 cells evaluated per slide. Two slides were made from each ejaculate. Motility characteristics that were evaluated included lateral head displacement, beat cross frequency, path velocity, path linearity, path straightness, percentage of motile cells, and percentage of progressively motile cells. Sperm cell morphology was also evaluated. Canine spermatozoa maintained good overall motility (mean +/- SD, 73 +/- 9%) during the procedure. Mean sperm motility and morphology measurements differed significantly between dogs (P<0.01). There was no difference (P>0.05) between the mean measurements of different ejaculates for an individual dog, or for different slides made from the same ejaculate. Mean motility values for the 10 dogs are reported. There was a significant but not strong correlation (r=0.44) between the percentage of progressively motile sperm cells and the percentage of sperm cells with normal morphology.     

Aerodynamic versus physical size of spores: Measurement and implication for respiratory deposition

Different methods available for size measurements of fungal and actinomycete spores were compared for four fungal species ( Penicillium brevicompactum, Penicillium melinii, Cladosporium cladosporioides , and Aspergillus versicolor ) and two actinomycete species ( Streptomyces albus and Thermoactinomyces vulgaris ). The physical size of spores was measured with three microscopic methods: with an optical microscope from stained (wet) slides, with an optical microscope from unstained (dry) slides and with an environmental scanning electron microscope (SEM) directly from the microbial culture. The aerodynamic diameter, d a , of airborne spores was measured with an aerodynamic particle sizer. The respiratory deposition of spores was calculated with a computer-based model. The environmental SEM measurements indicated larger size for fungal spores than the optical microscope, whereas for actinomycete spores, both microscopes gave comparable results. Optical microscopic measurements showed that the stained fungal spores were 1.1-1.2 times larger than the unstained ones, which was attributed to the different hydration status of spores. There was no clear trend in the relationship between the d a and the physical diameter measured with any of three tested microscopic methods. For example, the physical diameter of Cladosporium cladosporioides spores was larger than the d a by a factor ranging from 2.0 to 2.2, whereas the d a of Streptomyces albus spores was larger than the physical diameter by a factor of 1.3-1.5. Thus, the aerodynamic diameter of microbial spores cannot be accurately estimated solely based on the physical diameter but needs information on the density of the spores that may vary considerably. The results on the spore size were utilized to calculate respiratory deposition of spores. The errors in the size measurement were found to result in overestimation of the respiratory deposition of C. cladosporioides spores by a factor of 1.2-1.8, and underestimation of the respiratory deposition of S. albus spores by a factor of 0.6-0.7. These errors in the size measurement cause bias in the exposure assessment and in the estimation of the efficiency of control devices. More research is needed to standardize the method for particle diameter estimates applicable for airborne spores.     

Feasibility study on automated recognition of allergenic pollen: grass, birch and mugwort

Quantification of airborne pollen is an important tool in scientific research and patient care in allergy. The currently available method relies on microscopic examination of pollen slides, performed by qualified researchers. Although highly reliable, the method is labor intensive and requires extensive training of the researchers involved. In an approach to develop alternative detection methods, we performed a feasibility study on the automated recognition of the allergenic relevant pollen, grass, birch, and mugwort, by utilizing digital image analysis and pattern recognition tools. Of a total of 254 pollen samples (including 79 of grass, 79 of birch and 96 of mugwort), 97.2% were recognized correctly. This encouraging result provides a promising prospect for future developments.     

Identification of mycobacteria in peat moss processing plants: application of molecular biology approaches

Peat moss processing plant workers are exposed to high concentrations of bioaerosols. Although mycobacteria have been cultured from peat moss, no study has examined the workers' exposure to mycobacterial bioaerosols. We evaluated the presence of mycobacteria in air samples from peat moss processing plants using molecular biology approaches (cloning-sequencing and polymerase chain reaction (PCR)) and the workers exposure using immunoglobulin G (IgG) complexes to mycobacteria. In addition, species detected in air samples and in peat moss were compared. Two peat moss processing plants were chosen among 14 previously studied. A total of 49 clones were sequenced. Real-time PCR was also performed on the same air samples to evaluate the airborne concentration of mycobacteria and estimate exposure levels. Several Mycobacterium species were present in the air samples (M. malmoense, M. smegmatis, M. graceum, M. bohemicum, and M. interjectum). Mycobacterium avium was recovered by culture in peat moss but not in the air using the molecular approach. Total airborne Mycobacterium concentration was estimated at 8.2 x 10(8)/m3. Workers had IgG against the mycobacterial mix and M. avium, suggesting significant exposure. The findings from air samples, supported by IgG measurements, demonstrate that peat moss processing plant workers are exposed to mycobacteria in addition to other biological agents.     

Long-term trends and regional variation in the aeroallergen Alternaria in Cardiff and Derby UK – are differences in climate and cereal production having an effect?

Alternaria is a known aeroallergen, beinga risk factor in childhood and adult asthma.This study compared 1970–1996 daily records ofAlternaria spores in Cardiff and Derby,two very different regions of the UK. There hasbeen a dramatic upward trend in the seasonaltotal for Derby Alternariaspores whereas in Cardiff the trend isdownwards. On certain days in recent yearsDerby Alternaria spore counts haveexceeded 1000 spores per cubic metre of air,but in Cardiff such very high counts have notoccurred since 1970. Derby Alternariaspore levels were most positively correlatedwith wind from the SE (over large stretches ofarable land), whereas in Cardiff the mostpositive correlation was with wind from the north(over grassland). In Derby the increase incereal production, together with highermidsummer temperatures, could account for therising Alternaria counts. This upwardtrend in Derby is expected to continue whereasin Cardiff the coastal position together withthe small amounts of arable production ensureAlternaria spore levels will remain low.The comparison between these two siteshighlights regional differences in the numbersof airborne Alternaria spores andconfirms the importance of maintaining longestablished sites. In Derby these results haveimplications for Alternaria sensitivepatients as very high counts could trigger anasthma attack.     

Statistical significance analysis of longitudinal gene expression data

MOTIVATION: Time-course microarray experiments are designed to study biological processes in a temporal fashion. Longitudinal gene expression data arise when biological samples taken from the same subject at different time points are used to measure the gene expression levels. It has been observed that the gene expression patterns of samples of a given tumor measured at different time points are likely to be much more similar to each other than are the expression patterns of tumor samples of the same type taken from different subjects. In statistics, this phenomenon is called the within-subject correlation of repeated measurements on the same subject, and the resulting data are called longitudinal data. It is well known in other applications that valid statistical analyses have to appropriately take account of the possible within-subject correlation in longitudinal data. RESULTS: We apply estimating equation techniques to construct a robust statistic, which is a variant of the robust Wald statistic and accounts for the potential within-subject correlation of longitudinal gene expression data, to detect genes with temporal changes in expression. We associate significance levels to the proposed statistic by either incorporating the idea of the significance analysis of microarrays method or using the mixture model method to identify significant genes. The utility of the statistic is demonstrated by applying it to an important study of osteoblast lineage-specific differentiation. Using simulated data, we also show pitfalls in drawing statistical inference when the within-subject correlation in longitudinal gene expression data is ignored.     

The Environmental Impacts of Consumption at a Subnational Level

This article analyzes the environmental effects of resource consumption at a subnational level (by Cardiff, the capital city of Wales), using the Ecological Footprint as a measure of impact assessment. The article begins by providing a short critique of the Footprint methodology and the limitations of methods traditionally used to calculate national Footprint accounts. We then describe the Footprint methodology developed by the Stockholm Environment Institute to overcome some of these problems and used as the basis of the Reducing Wales' Ecological Footprint project, of which the Cardiff study has been a part. The main portion of this article focuses on presenting and discussing the Footprint results for Cardiff. The Ecological Footprint of household consumption in Cardiff will be presented using the international Classification of Individual Consumption According to Purpose (COICOP). Based on the results, we found that the areas of consumption that are a priority for Cardiff in terms of reducing resource use are food and drink, passenger transport (car and aviation), domestic fuel consumption, waste, and tourism. We also discuss how these findings have been presented to the Cardiff Council. We report on the initial reactions of policy officers to the Footprint results and how the Council plans to use them to influence policy decisions relating to sustainability. Finally, in the Conclusions section, we briefly explain the value of applying the Ecological Footprint at a subnational level and its value as an evidence-based tool for sustainability decision making.