Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.     

Risk Assessment Modeling: Beyond Exposure and Effects

Problem formulation, risk analysis, and risk characterization are, respectively, the design, estimation, and interpretation stages of risk assessment. Models traditionally have been used to estimate exposure and effects; now opportunities are growing to use them to design and interpret risk assessments as well. This could raise the level of rigor, reproducibility, and transparency in the risk assessment process, and improve the way information and expertise gets integrated to advise risk managers. The importance of good design and interpretation to the success of risk assessment and risk management, and the role of modeling in that success, is becoming increasingly apparent, but to date models are used only to a fraction of their potential. We provide two examples of the use of models to design and interpret risk assessments. The first looks at the use of models to better characterize risks by modeling uncertainties and exposure from offsite sources, and the second to forecast future risks of a new technology. Following the examples, we discuss some important obstacles to translating new modeling opportunities into practice. These include practical limits on the abilities of organizations to assimilate new tools and methods, and conceptual limits in the way people think about models.     

Evaluation of the Limulus Amebocyte Lysate and Recombinant Factor C Assays for Assessment of Airborne Endotoxin

As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) samplers, Button samplers, closed-face cassettes, and cyclone samplers. Field sampling was performed in 10 livestock production facilities, including those housing swine, chicken, turkey, dairy cows, cattle, and horses. Laboratory sampling was performed in exposure chambers using resuspended airborne dust collected in five livestock facilities. Paired samples were extracted in pyrogen-free water with 0.05% Tween 20 and analyzed using LAL and rFC assays. In 402 field sample pairs there was excellent agreement between endotoxin concentrations determined by LAL and rFC (r = 0.93; P < 0.0001). In 510 laboratory sample pairs there was also excellent agreement between the two assays (r = 0.86; P < 0.0001). Correlations for subgroups of facility or dust type ranged from 0.65 to 0.96. Mixed-model analysis of variance (ANOVA) for the field studies showed significant interactions of facility-sampler and facility-assay. rFC/LAL ratios of the geometric means were 0.9 to 1.14 for the samplers (not significantly different from 1.0). The data from this study demonstrate that the LAL assay and the rFC assay return similar estimates of exposure in livestock facilities. Both methods provided suitable lower limits of detection such that all but 19 of 1,824 samples were quantifiable.Endotoxin, or lipopolysaccharide, is a pathogen-associated molecular pattern of Gram-negative bacteria that associates with MD-2 (lymphocyte antigen 96) to act as a ligand for Toll-like receptor 4 (10). Through this process, inhaled endotoxin induces lung inflammation and can both increase neutrophilic asthma and decrease allergy and atopic asthma (2, 8, 9, 17, 26, 33). Endotoxin is an inflammatory component of most organic dusts (6). Exposure to endotoxins in agricultural dusts, including swine, poultry, and grain, has been associated with asthma, chronic bronchitis, organic dust toxic syndrome (toxic pneumonitis), chronic obstructive pulmonary disease, and declines in pulmonary function (5, 18, 24, 25, 35, 36, 38). High endotoxin exposure also occurs in municipal composting (37), seed and bulb handling (27), and wastewater treatment plants (13, 28), to name a few. In addition to occupational environments, endotoxin exposure in domestic environments is a risk factor for asthma (33), with high occupancy, poverty, pets, pests, and household cleanliness being the major predictors of exposure (32).The kinetic chromogenic Limulus amebocyte lysate (LAL) assay is the most widely used assay for endotoxin measurement for environmental samples (6). This assay uses an endotoxin-triggered enzyme cascade from the Atlantic horseshoe crab (Limulus polyphemus) to cleave a colorimetric substrate. Although the LAL assay is exquisitely sensitive, variability can arise from interlot variations in the Limulus lysate and differences in laboratory methods for sample collection, sample handling and storage, sample extraction, and sample analysis (7, 12, 14, 15, 19, 21, 29, 30, 34). In addition, some implementations of the LAL assay may experience interference from other molecules, such as fungal (1→3)-β-d-glucans (3, 22). A recombinant factor C (rFC) assay that uses rFC reagent produced from the cDNA of the Mangrove horseshoe crab (Cacinoscorpius rotundicauda) was recently developed (4). Since the rFC assay uses a recombinant reagent, the reactivity to endotoxins should vary less between lots than for the Limulus lysate.The goal of this study was to determine the comparability, across a wide range of endotoxin levels, of the kinetic chromogenic LAL and the fluorometric rFC assays for assessing airborne endotoxin. To accomplish this, we performed air sampling in 10 livestock environments and in chamber studies using resuspended dust collected from these livestock environments. Livestock environments are recognized as containing considerable amounts of airborne endotoxin arising from a variety of Gram-negative bacteria. Eight samples were collected simultaneously on a rotating mannequin using pairs of four types of commonly used samplers: two for inhalable dust (Institute of Occupational Medicine [IOM] and Button), one for total dust (closed-face cassette), and one for respirable dust (SKC aluminum cyclone). This resulted in 912 pairs of samples (total n = 1,824).     

不同保存方式下蝗虫组织DNA的提取及RAPD分析

为了开展蝗虫分子系统学研究,分别对冷冻、乙醇浸泡(100%、乙醇、70％乙醇)和干制蝗虫标本用饱和NaCl法进行了基因组DNA的提取,并用随机引物进行扩增,结果表明：70％乙醇固定的标本和部分干标本提取的总DNA得率较低,在琼脂糖凝胶电泳检测中大音琏分有明显降解,导致PCR扩增中信息缺失,甚至无扩增条带;而保存完好的干标本、-20℃冷冻标本和100%,乙醇浸泡标本提取的总DNA带型整齐,无拖尾,PCR扩增结果的稳定性好,成为蝗虫分子系统学研究中首选的三种保存方式。     

Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions,

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Highlights

• •Endogenous plasma/serum QC marker that quantifies cumulative sample thawed time.
• •Mechanism of marker change known, and rate law established.
• •Data interpretation based on documented population averages and rate law.
• •Blind-challenge verified; utility proven via exposure of undisclosed freezer outage.

     

Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols,Matrix Effects,and Nucleic Acid Interference

Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4′,6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over- or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference.     

Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.     

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

杨梅基因组DNA提取方法筛选和优化研究

选择具有代表性的杨梅品种20份,分别采用CTAB法和SDS法,选取嫩叶进行基因组DNA提取比较研究,结果表明,由于叶片中含有较高的酚类物质,相对来说CTAB法优于SDS法,提取的基因组DNA色泽白亮且数量多,而且同一提取方法如CTAB法,不同品种之间提取得率也有较大差异。在CTAB法的基础上,选取两个品种研究水浴时间对DNA提取的影响,发现DNA得率随水浴时间的延长呈现抛物线态势,最佳水浴时间随样品颗粒的大小而变化,一般最佳时间为30～40min,样品颗粒较细时水浴时间以30min为最佳,较粗时则为40min。添加异丙醇量以2/3为好,析出DNA的最佳离心速度和时间分别为5 000r/min和10～15min。     

Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however, the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.     

乳腺分型及乳腺厚度与全数字化乳腺X 线摄影曝光条件 及平均腺体剂量的关系

目的:探讨不同乳腺分型(Ⅰ型(脂肪型)、Ⅱ型(致密型)、Ⅲ型(中间型)、Ⅳ型(导管型))、不同乳腺厚度与全数字化乳腺X射线摄影曝光条件(kV、mAs)、平均腺体剂量(mGy)之间的关系。方法:回顾性分析2009年9月~2010年6月间采用德国Siemens公司MAMMOMAT Novation DR全数字化乳腺摄影系统、自动曝光控制模式下摄影所获得的2 000例头尾位和内外侧斜位乳腺片,分析7 840幅Ⅰ级乳腺照片中不同乳腺分型、不同乳腺厚度的曝光条件、平均腺体剂量,以研究乳腺分型及乳腺厚度与全数字化乳腺X线摄影曝光条件及平均腺体剂量的关系。结果:当乳腺厚度相同时,Ⅱ型(致密型)乳腺的曝光条件及平均腺体剂量最大,Ⅳ型(导管型)次之,Ⅲ型(中间型)再次之,Ⅰ型(脂肪型)乳腺的曝光条件及平均腺体剂量最小。无论何种乳腺分型,随着乳腺厚度的增加,全数字化乳腺X射线摄影曝光条件及平均腺体剂量随之增加。结论:乳腺分型及乳腺厚度与全数字化乳腺X射线摄影曝光条件及平均腺体剂量关系密切,乳腺腺体组织越致密、厚度越厚,其曝光条件及平均腺体剂量就越大。     

乳腺分型及乳腺厚度与全数字化乳腺X线摄影曝光条件及平均腺体剂量的关系

目的：探讨不同乳腺分型（Ⅰ型（脂肪型）、Ⅱ型（致密型）、Ⅲ型（中间型）、Ⅳ型（导管型））、不同乳腺厚度与全数字化乳腺X射线摄影曝光条件（kV、mms）、平均腺体剂量（mGy）之间的关系。方法：回顾性分析2009年9月-2010年6月间采用德国Siemens公司MAMMOMAT Novation DR全数字化乳腺摄影系统、自动曝光控制模式下摄影所获得的2000例头尾位和内外侧斜位乳腺片,分析7840幅Ⅰ级乳腺照片中不同乳腺分型、不同乳腺厚度的曝光条件、平均腺体剂量,以研究乳腺分型及乳腺厚度与全数字化乳腺X线摄影曝光条件及平均腺体剂量的关系。结果：当乳腺厚度相同时,Ⅱ型（致密型）乳腺的曝光条件及平均腺体剂量最大,Ⅳ型（导管型）次之,Ⅲ型（中间型）再次之,Ⅰ型（脂肪型）乳腺的曝光条件及平均腺体剂量最小。无论何种乳腺分型,随着乳腺厚度的增加,全数字化乳腺X射线摄影曝光条件及平均腺体剂量随之增加。结论：乳腺分型及乳腺厚度与全数字化乳腺X射线摄影曝光条件及平均腺体剂量关系密切,乳腺腺体组织越致密、厚度越厚,其曝光条件及平均腺体剂量就越大。     

蝴蝶兰叶片蛋白质提取及双向电泳体系优化

通过对蛋白质提取、IPG胶条选择、上样量、水化方式、聚焦条件等方面的优化,建立蝴蝶兰叶片蛋白质的双向电泳体系。结果表明,采用酚抽提法提取蝴蝶兰叶片蛋白质的纯度较高,复溶较完全;双向电泳优化体系选用24 cm pH 3～10 NL的IPG胶条,被动水化,上样量为1.35 mg,B1程序进行等电聚焦,12%分离胶进行第二向电泳,考马斯亮蓝G-250染色。该方法获得分辨率较高、重复性较好的蝴蝶兰叶片双向电泳图谱,蛋白数点多达1163个,可以满足蝴蝶兰蛋白质组学研究和分析。     

米心水青冈基因组DNA提取及RAPD反应体系优化

采用4种方法对米心水青冈基因组DNA进行提取,通过比较得出改良CTAB法提出的DNA纯度较高,能够达到扩增要求,因此采用此方法用于正式DNA的提取。适合米心水青冈的RAPD反应体系为：反应体积为25 μL,模板DNA40 ng,引物0.8 μmol·L^-1,Taq聚合酶1.25 U,Mg²⁺浓度2.0 mmol·L^-1,dNTP浓度0.16 mmol·L^-1。适合米心水青冈RAPD扩增程序： 94℃预变性3 min,一个循环,94℃变性30 s,37℃退火1 min,72℃延伸2 min,45个循环。     

Quantification of Soil-to-Plant Transport of Recombinant Nucleopolyhedrovirus: Effects of Soil Type and Moisture, Air Currents, and Precipitation

Significantly more occlusion bodies (OB) of DuPont viral construct HzSNPV-LqhIT2, expressing a scorpion toxin, were transported by artificial rainfall to cotton plants from sandy soil (70:15:15 sand-silt-clay) than from silt (15:70:15) and significantly more from silt than from clay (15:15:70). The amounts transported by 5 versus 50 mm of precipitation were the same, and transport was zero when there was no precipitation. In treatments that included precipitation, the mean number of viable OB transported to entire, 25- to 35-cm-tall cotton plants ranged from 56 (clay soil, 5 mm of rain) to 226 (sandy soil, 50 mm of rain) OB/plant. In a second experiment, viral transport increased with increasing wind velocity (0, 16, and 31 km/h) and was greater in dry (−1.0 bar of matric potential) than in moist (−0.5 bar) soil. Wind transport was greater for virus in a clay soil than in silt or sand. Only 3.3 × 10⁻⁷ (clay soil, 5 mm rain) to 1.3 × 10⁻⁶ (sandy soil, 50 mm rain) of the OB in surrounding soil in experiment 1 or 1.1 × 10⁻⁷ (−0.5 bar sandy soil, 16-km/h wind) to 1.3 × 10⁻⁶ (−1.0 bar clay soil, 31-km/h wind) in experiment 2 were transported by rainfall or wind to cotton plants. This reduces the risk of environmental release of a recombinant nucleopolyhedrovirus (NPV), because only a very small proportion of recombinant virus in the soil reservoir is transported to vegetation, where it can be ingested by and replicate in new host insects.     

钝节拟丽藻抑菌作用及提取条件的优化

研究了钝节拟丽藻提取液的抑菌作用,优化了提取条件。结果表明,钝节拟丽藻乙醇提取液对金黄色葡萄球菌、枯草杆菌有明显抑制作用,对大肠杆菌、变形杆菌、酿酒酵母和黄曲霉无明显抑制作用。通过抑菌圈直径方法,对影响钝节拟丽藻抑菌物质提取的条件进行了单因素试验。正交试验中的固液比和提取温度影响样品提取液对金黄色葡萄球菌和枯草杆菌的抑菌效果,作用极显著,其交互作用对金黄色葡萄球菌和枯草杆菌的抑菌效果作用显著。最佳提取条件为固液比1∶20,提取温度85℃,50%乙醇溶剂,回流提取6 h。     

杏鲍菇菌丝体水溶性多糖提取及培养条件优化

以马铃薯葡萄糖综合培养基(PDP)为基础培养基,采用正交试验法优化杏鲍菇菌丝体多糖发酵条件,对接种量、摇床转速和培养时间等因素对多糖含量的影响进行了研究。采用水提醇沉法提取多糖,苯酚—浓硫酸法进行多糖含量测定。结果表明,最佳培养条件:接种量为每瓶1块直径为1cm的菌块,转速为140r/min,培养时间为8d。此时杏鲍菇菌丝体多糖含量最高,为75.1mg/g。     

Assessment of Field Reentry Exposure to Pesticides: Limitations,Uncertainties, and Alternatives

There are many complex scientific as well as regulatory issues associated with the assessment of field reentry exposure to pesticides. These issues largely come from the limitations and uncertainties inherent in the simplified algorithm that many regulatory agencies use to estimate the reentry exposure. The main objective of this Perspective Article is to bring out these issues systematically in an open forum for further consideration by the exposure assessment community. Accordingly, in this Perspective Article, the simplified algorithm is elaborated first to provide a basic understanding for the complex issues involved. Both the limitations and the uncertainties revolving around this algorithm's real-time application are then discussed extensively, including those specific to monitoring dermal residues in a study trial, measuring dislodgeable foliar residues, and dealing with dislodgeability as well as transferability of foliar residues. The discussion ends by proposing a more practical alternative to the current assessment approach.