Increased levels of markers of microbial exposure in homes with indoor storage of organic household waste

As part of environmental management policies in Europe, separate collection of organic household waste and nonorganic household waste has become increasingly common. As waste is often stored indoors, this policy might increase microbial exposure in the home environment. In this study we evaluated the association between indoor storage of organic waste and levels of microbial agents in house dust. The levels of bacterial endotoxins, mold beta(1-->3)-glucans, and fungal extracellular polysaccharides (EPS) of Aspergillus and Penicillium species were determined in house dust extracts as markers of microbial exposure. House dust samples were collected in 99 homes in The Netherlands selected on the basis of whether separated organic waste was present in the house. In homes in which separated organic waste was stored indoors for 1 week or more the levels of endotoxin, EPS, and glucan were 3.2-, 7.6-, and 4. 6-fold higher, respectively (all P < 0.05), on both living room and kitchen floors than the levels in homes in which only nonorganic residual waste was stored indoors. Increased levels of endotoxin and EPS were observed, 2.6- and 2.1-fold (P < 0.1), respectively, when separated organic waste was stored indoors for 1 week or less, whereas storage of nonseparated waste indoors had no effect on microbial agent levels (P > 0.2). The presence of textile floor covering was another major determinant of microbial levels (P < 0.05). Our results indicate that increased microbial contaminant levels in homes are associated with indoor storage of separated organic waste. These increased levels might increase the risk of bioaerosol-related respiratory symptoms in susceptible people.     

Evaluation of a Low-Cost Electrostatic Dust Fall Collector for Indoor Air Endotoxin Exposure Assessment

Exposure to endotoxin in home environments has become a key issue in asthma and allergy research. Most studies have analyzed floor or mattress dust endotoxin, but its validity as a proxy for airborne exposure is unknown, while active airborne dust sampling is not feasible in large-scale population studies because of logistic and financial limitations. We therefore developed and evaluated a simple passive airborne dust collection method for airborne endotoxin exposure assessment. We explored an electrostatic dust fall collector (EDC), consisting of a 42- by 29.6-cm-sized folder with four electrostatic cloths exposed to the air. The EDC was tested during two 14-day periods in seven nonfarm and nine farm homes and in farm stables. In parallel, active airborne dust sampling was performed with Harvard impactors and floor dust collected by vacuuming, using nylon sampling socks. The endotoxin levels could be measured in all EDC cloth extracts. The levels (in EU/m²) between EDCs used simultaneously or in different sampling periods in the same home correlated strongly (r > 0.8). EDC endotoxin also correlated moderately to strongly (r = 0.6 to 0.8) with the endotoxin measured by active airborne dust sampling and living room floor dust sampling and—in farm homes—with the endotoxin captured by the EDC in stables. In contrast, endotoxin levels measured by floor dust sampling showed only a poor correlation with the levels measured by active airborne dust sampling. We therefore conclude that measuring endotoxin levels with the EDC is a valid measure of average airborne endotoxin exposure, while reproducibility over time is at least equivalent to that of reservoir dust analyses.     

Dynamics of Soil Organic Carbon and Aggregate Stability with Grazing Exclusion in the Inner Mongolian Grasslands

Grazing exclusion (GE) has been deemed as an important approach to enhance the soil carbon storage of semiarid grasslands in China; however, it remains unclear how different organic carbon (OC) components in soils vary with the duration of GE. Here, we observed the changing trends of different OC components in soils with increased GE duration in five grassland succession series plots, ranging from free grazing to 31-year GE. Specifically, we measured microbial biomass carbon (MBC), easily oxidizable OC (EOC), water-soluble OC (WSOC), and OC in water stable aggregates (macroaggregates [250–2000 μm], microaggregates [53–250 μm], and mineral fraction [< 53 μm]) at 0–20 cm soil depths. The results showed that GE significantly enhanced EOC and WSOC contents in soils, but caused a decline of MBC at the three decade scale. Macroaggregate content (F = 425.8, P < 0.001), OC stored in macroaggregates (F = 84.1, P < 0.001), and the mean weight diameter (MWD) of soil aggregates (F = 371.3, P < 0.001) increased linearly with increasing GE duration. These findings indicate that OC stored in soil increases under three-decade GE with soil organic matter (SOM) stability improving to some extent. Long-term GE practices enhance the formation of soil aggregates through higher SOM input and an exclusion of animal trampling. Therefore, the practice of GE may be further encouraged to realize the soil carbon sequestration potential of semi-arid grasslands, China.     

Seasonal Variations of Indoor Microbial Exposures and Their Relation to Temperature,Relative Humidity,and Air Exchange Rate

Indoor microbial exposure has been related to adverse pulmonary health effects. Exposure assessment is not standardized, and various factors may affect the measured exposure. The aim of this study was to investigate the seasonal variation of selected microbial exposures and their associations with temperature, relative humidity, and air exchange rates in Danish homes. Airborne inhalable dust was sampled in five Danish homes throughout the four seasons of 1 year (indoors, n = 127; outdoors, n = 37). Measurements included culturable fungi and bacteria, endotoxin, N-acetyl-beta-d-glucosaminidase, total inflammatory potential, particles (0.75 to 15 μm), temperature, relative humidity, and air exchange rates. Significant seasonal variation was found for all indoor microbial exposures, excluding endotoxin. Indoor fungi peaked in summer (median, 235 CFU/m³) and were lowest in winter (median, 26 CFU/m³). Indoor bacteria peaked in spring (median, 2,165 CFU/m³) and were lowest in summer (median, 240 CFU/m³). Concentrations of fungi were predominately higher outdoors than indoors, whereas bacteria, endotoxin, and inhalable dust concentrations were highest indoors. Bacteria and endotoxin correlated with the mass of inhalable dust and number of particles. Temperature and air exchange rates were positively associated with fungi and N-acetyl-beta-d-glucosaminidase and negatively with bacteria and the total inflammatory potential. Although temperature, relative humidity, and air exchange rates were significantly associated with several indoor microbial exposures, they could not fully explain the observed seasonal variations when tested in a mixed statistical model. In conclusion, the season significantly affects indoor microbial exposures, which are influenced by temperature, relative humidity, and air exchange rates.     

Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.     

Seasonal Dynamics of Shallow-Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R² = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R² = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the α, β, and γ-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.     

Characterization of Lipopolysaccharides Present in Settled House Dust

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C_16:0 and C_18:0) 3-OHFAs were predominant in HD compared with short-chain (C_10:0, C_12:0, and C_14:0) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C_16:0 was negatively correlated and 3-OH C_18:0 was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C_10:0, the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.     

Vascular Endothelial Cell Injury Is an Important Factor in the Development of Encapsulating Peritoneal Sclerosis in Long-Term Peritoneal Dialysis Patients

MethodsEighty-three peritoneal membrane samples taken at catheter removal were examined to identify pathological characteristics of chronic peritoneal deterioration, which promotes EPS in patients undergoing long-term PD treatment with low occurrence of peritonitis.ResultsAccording to univariable logistic regression analysis of the pathological findings, thickness of the peritoneal membrane (P = 0.045), new membrane formation score (P = 0.006), ratio of luminal diameter to vessel diameter (L/V ratio, P<0.001), presence of CD31-negative vessels (P = 0.021), fibrin deposition (P<0.001), and collagen volume fraction (P = 0.018) were associated with EPS development. In analyses of samples with and without EPS matched for PD treatment period, non-diabetes, and PD solution, univariable analysis identified L/V ratio (per 0.1 increase: odds ratio (OR) 0.44, P = 0.003) and fibrin deposition (OR 6.35, P = 0.027) as the factors associated with EPS. L/V ratio was lower in patients with fibrin exudation than in patients without fibrin exudation.ConclusionsThese findings suggest that damage to vascular endothelial cells, as represented by low L/V ratio, could be a predictive finding for the development of EPS, particularly in long-term PD patients unaffected by peritonitis.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

Rapid Detection of Meat Spoilage by Measuring Volatile Organic Compounds by Using Proton Transfer Reaction Mass Spectrometry

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4°C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.     

EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10⁻⁷) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10⁻⁵). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.     

Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System

The objective of this study was to evaluate the effects of partially replacing dry ground corn with glycerin on ruminal fermentation using a dual-flow continuous culture system. Six fermenters (1,223 ± 21 ml) were used in a replicated 3x3 Latin square arrangement with three periods of 10 d each, with 7 d for diet adaptation and 3 d for sample collections. All diets contained 75% concentrate and three dietary glycerin levels (0, 15, and 30% on dry matter basis), totaling six replicates per treatment. Fermenters were fed 72 g of dry matter/d equally divided in two meals/d, at 0800 and 2000 h. Solid and liquid dilution rates were adjusted daily to 5.5 and 11%/h, respectively. On d 8, 9, and 10, samples of 500 ml of solid and liquid digesta effluent were mixed, homogenized, and stored at -20°C. Subsamples of 10 ml were collected and preserved with 0.2 mL of a 50% H₂SO₄ solution for later determination of NH₃-N and volatile fatty acids. Microbial biomass was isolated from fermenters for chemical analysis at the end of each experimental period. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Glycerin levels did not affect apparent digestibility of DM (P _Lin. = 0.13; P _Quad. = 0.40), OM (P _Lin. = 0.72; P _Quad. = 0.15), NDF (P _Lin. = 0.38; P _Quad. = 0.50) and ADF (P _Lin. = 0.91; P _Quad. = 0.18). Also, glycerin inclusion did not affect true digestibility of DM (P _Lin. = 0.35; P _Quad. = 0.48), and OM (P _Lin. = 0.08; P _Quad. = 0.19). Concentrations of propionate (P < 0.01) and total volatile fatty acids (P < 0.01) increased linearly and concentrations of acetate (P < 0.01), butyrate (P = 0.01), iso-valerate (P < 0.01), and total branched-chain volatile fatty acids, as well as the acetate: propionate ratio (P < 0.01) decreased with glycerin inclusion. Linear increases on NH₃-N concentration in digesta effluent (P < 0.01) and on NH₃-N flow (P < 0.01) were observed due to glycerin inclusion in the diets. Crude protein digestibility (P = 0.04) and microbial N flow (P = 0.04) were greater in the control treatment compared with the other treatments and responded quadratically with glycerin inclusion. Furthermore, the inclusion of glycerin linearly decreased (P = 0.02) non-ammonia N flow. Glycerin levels did not affect the flows of total N (P _Lin. = 0.79; P _Quad. = 0.35), and dietary N (P _Lin. = 0.99; P _Quad. = 0.07), as well as microbial efficiency (P _Lin. = 0.09; P _Quad. = 0.07). These results suggest that partially replacing dry ground corn with glycerin may change ruminal fermentation, by increasing total volatile fatty acids, and propionate concentration without affecting microbial efficiency, which may improve glucogenic potential of beef cattle diets.     

Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10⁶ to 10⁷ CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Mitochondrial Regulation of NADPH Oxidase in Hindlimb Unweighting Rat Cerebral Arteries

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.     

Environmental Determinants of and Impact on Childhood Asthma by the Bacterial Community in Household Dust

Asthma increased dramatically in the last decades of the 20th century and is representative of chronic diseases that have been linked to altered microbial exposure and immune responses. Here we evaluate the effects of environmental exposures typically associated with asthma protection or risk on the microbial community structure of household dust (dogs, cats, and day care). PCR-denaturing gradient gel analysis (PCR-DGGE) demonstrated that the bacterial community structure in house dust is significantly impacted by the presence of dogs or cats in the home (P = 0.0190 and 0.0029, respectively) and by whether or not children attend day care (P = 0.0037). In addition, significant differences in the dust bacterial community were associated with asthma outcomes in young children, including wheezing (P = 0.0103) and specific IgE (P = 0.0184). Our findings suggest that specific bacterial populations within the community are associated with either risk or protection from asthma.Recent studies have begun to explore the microbial composition of house dust, finding great diversity and a high abundance of Gram-positive organisms (18, 24). Yet this transient community remains relatively unexplored despite increasing evidence of an association between microbial exposure and human health, in particular, the development of chronic diseases such as asthma. Settings with high levels of microbial exposure, such as farms and day care centers, have been associated with protection from asthma (3, 23), while interventions that reduce home microbial exposures have been related to higher rates of allergic sensitization (29). Moreover, variability in home microbial exposures has been linked to differences in immune response and asthma risk in childhood (1, 4). Finally, genes involved in innate immune responses to microbes have been found to interact with microbial exposures, resulting in altered risks for asthma (10). Microbial exposure has primarily been assessed in these studies through measurement of surrogates, such as lipopolysaccharide (LPS, or endotoxin), a component of the outer membrane of Gram-negative bacteria (16), and more recently N-acetyl muramic acid for Gram-positive bacteria (28) and ergosterol for fungi (25). While the use of such surrogates has confirmed a negative relationship between levels of microbial exposure and the development of asthma, exploration is needed beyond a handful of surrogates to more completely represent the complexity of the actual microbial communities present in homes.A preliminary experiment was performed to characterize the bacterial communities of dust from homes in Tucson, AZ. First, general bacterial community diversity was examined in house dust samples by using a PhyloChip microarray as described by Brodie et al. (5). These samples were obtained from homes of four volunteers in the Tucson area. Array results revealed an average of 295 ± 16 (mean ± standard deviation) unique operational taxonomic units (OTUs) per sample, where a taxon was considered present in the sample when more than 92% of the assigned probe pairs for its corresponding probe set were positive. Of these OTUs, 85% belonged to the same 13 divisions in each dust sample, with the 5 most dominant divisions including the Alphaproteobacteria (17.6 ± 2.6%), Firmicutes (16.3 ± 4.1%), Actinobacteria (13.4 ± 2.2%), Gammaproteobacteria (11.8 ± 1.1%), and Deltaproteobacteria (8.7 ± 0.4%) (see Fig. S1 in the supplemental material). Distributions among the 13 major divisions were fairly consistent but not identical among the four different dust samples. The remaining 15% of OTUs belonged to 33 other bacterial divisions but were present at frequencies of less than 1% each. Thus, the number of OTUs and the phylogenetic distributions of these OTUs among the four households were similar.A second experiment was performed to see how the structure of house dust communities varies with environmental parameters and asthma outcomes. The dust samples, examined using denaturing gradient gel electrophoresis (DGGE), were obtained from homes of infants enrolled in the Infant Immune Study (IIS), a nonselected birth cohort (12). Dust samples were collected when the child was approximately 3 months old, at which time the home condition was evaluated. Other information available on this cohort includes data on (i) early life exposures potentially related to asthma risk (the presence of a dog or cat in the home and day care attendance in the first months of life) and (ii) asthma-related outcomes of wheezing and aeroallergen sensitization (specific IgE [Sp-IgE]) (7). A subset (n = 44) of the IIS dust samples was used in this analysis. Based upon the premise that health outcomes will be influenced by the most abundant members of the dust bacterial community (in terms of numbers), DGGE of 16S rRNA gene PCR amplicons was selected as the profile technique to compare the microbial community structures of dust samples from the 44 homes. PCR-DGGE profiles evaluate 16S rRNA genes from all bacterial populations whose initial template concentrations represent >1% of the total community DNA (6, 13, 17). PhyloChip analysis was not used for this experiment because the cost would have been prohibitive for the number of households analyzed.DNA extraction was performed on the IIS dust samples by direct lysis using the Fast DNA spin kit for soil (MP Biomedicals, Solon, OH) as described previously (9). The amount of dust used for extraction ranged from 21 to 35 mg. For each set of extractions a DNA blank (no dust) was included to monitor potential contamination during the extraction process. The extracted DNA was labeled with the PicoGreen double-stranded DNA (dsDNA) quantitation reagent (Molecular Probes, Eugene, OR) and quantified using the Turner Biosystems TBS 380 fluorometer. DNA yields ranged from 0.2 to 41.2 ng DNA/mg of dust.For DGGE analysis, the community DNA obtained from the dust samples was PCR amplified (25 cycles) using the V7/V8 variable region of the 16S rRNA gene and primers 1070f and 1392R with a 40-bp GC clamp (11), following the protocol of Colores et al. (8). Unacetylated bovine serum albumin (Sigma, St. Louis, MO) was added to the PCR mixture at a concentration of 0.4 g liter⁻¹ to relieve PCR inhibition. Amplicons were separated on DGGE gels by using the Bio-Rad Laboratories system (D-Code; Hercules, CA) with a 6% acrylamide gel and a 45% to 65% urea-formamide denaturing gradient. Each DGGE gel run in this study examined either an environmental exposure or an asthma-related outcome variable, as detailed in Tables ​Tables11 and ​and2.2. For example, children reported to have actively wheezed on at least two out of three questionnaires obtained at 1, 2, and 3 years were compared with children for whom all three questionnaires were completed but for whom no wheezing was reported. Fourteen samples were analyzed on each gel, including seven different households from the negative group and seven different households from the positive group. Two lanes of the gel were also loaded with negative controls. DGGE gels were run for 15.5 h at a constant voltage of 50 V, stained with 3× SYBR green I (Molecular Probes, Eugene, OR) for 40 min, and then visualized.

TABLE 1.

Gel comparison groups for early life environmental exposures

Further Insights into Metal-DOM Interaction: Consideration of Both Fluorescent and Non-Fluorescent Substances

Information on metal binding with fluorescent substances has been widely studied. By contrast, information on metal binding with non-fluorescent substances remains lacking despite the dominance of these substances in aquatic systems. In this study, the metal binding properties of both fluorescent and non-fluorescent substances were investigated by using metal titration combined with two-dimensional correlation spectroscopy (2D–COS) analysis. The organic matters in the eutrophic algae-rich lake, including natural organic matters (NOM) and algae-induced extracellular polymeric substances (EPS), both contained fluorescent and non-fluorescent substances. The peaks in the one-dimensional spectra strongly overlapped, while 2D–COS can decompose the overlapped peaks and thus enhanced the spectral resolution. Moreover, 2D FTIR COS demonstrated that the binding susceptibility of organic ligands in both NOM and algal EPS matrices followed the order: 3400>1380>1650 cm⁻¹, indicative the significant contribution of non-fluorescent ligands in metal binding. The modified Stern-Volmer equation also revealed a substantial metal binding potential for the non-fluorescent substances (logK_M: 3.57∼4.92). As for the effects of organic ligands on metal binding, EPS was characterized with higher binding ability than NOM for both fluorescent and non-fluorescent ligands. Algae-induced EPS and the non-fluorescent substances in eutrophic algae-rich lakes should not be overlooked because of their high metal binding potential.     

The ecology of microscopic life in household dust

We spend the majority of our lives indoors; yet, we currently lack a comprehensive understanding of how the microbial communities found in homes vary across broad geographical regions and what factors are most important in shaping the types of microorganisms found inside homes. Here, we investigated the fungal and bacterial communities found in settled dust collected from inside and outside approximately 1200 homes located across the continental US, homes that represent a broad range of home designs and span many climatic zones. Indoor and outdoor dust samples harboured distinct microbial communities, but these differences were larger for bacteria than for fungi with most indoor fungi originating outside the home. Indoor fungal communities and the distribution of potential allergens varied predictably across climate and geographical regions; where you live determines what fungi live with you inside your home. By contrast, bacterial communities in indoor dust were more strongly influenced by the number and types of occupants living in the homes. In particular, the female : male ratio and whether a house had pets had a significant influence on the types of bacteria found inside our homes highlighting that who you live with determines what bacteria are found inside your home.     

Effects of Acid on Plant Litter Decomposition in an Arctic Lake

The effects of acid on the microbial decomposition of the dominant aquatic macrophyte (Carex sp.) in Toolik Lake, Alaska were studied in microcosms during the ice-free season of 1980. Toolik Lake is slightly buffered, deep, and very oligotrophic. Microbial activities, as determined by ¹⁴C-acetate incorporation into extractable lipids, associated with Carex litter were significantly (P < 0.01) reduced within 2 days at pHs of 3.0 and 4.0, but not 5.0, 5.5, or 6.0, as compared with ambient controls (pH 7.4). ATP levels were significantly reduced at pH 3.0, but not at the other pHs tested. After 18 days, microbial activity significantly correlated with weight loss (P < 0.05), nitrogen content (P < 0.01), and C/N ratios (P < 0.01) of the litter, but did not correlate with ATP levels. Scanning electron microscopy of the litter surface revealed that the fungi present at ambient pH did not become dominant at pHs below 5.5, diatoms were absent below pH 4.0, and bacterial numbers and extracellular slime were greatly reduced at pH 4.0 and below. Mineralization of Carex¹⁴C-lignin-labeled or ¹⁴C-cellulose-labeled lignocellulose was reduced at pH 2.0, but not at pH 4.0, 5.0, or 6.0, compared with controls (pH 7). We concluded that if the pH of the water from this slightly buffered lake was sufficiently reduced, rates of litter decomposition would be significantly reduced.     

Hydroxyurea affects in vitro porcine oocyte maturation through increased apoptosis and oxidative stress

Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus–oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.