Environmental microbial contamination in a stem cell bank

AIM: The aim of this study was to evaluate the main environmental microbial contaminants of the clean rooms in our stem cell bank. METHODS AND RESULTS: We have measured the microbial air contamination by both passive and active air sampling and the microbial monitoring of surfaces by means of Rodac plates. The environmental monitoring tests were carried out in accordance with the guidelines of European Pharmacopeia and US Pharmacopeia. The micro-organisms were identified by means of an automated system (VITEK 2). During the monitoring, the clean rooms are continually under good manufacturing practices specifications. The most frequent contaminants were Gram-positive cocci. CONCLUSIONS: The main contaminants in our stem cell bank were coagulase-negative staphylococci and other opportunistic human pathogens. In order to assure the levels of potential contamination in both embryonic and adult stem cell lines, a continuous sampling of air particles and testing for viable microbiological contamination is necessary. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first evaluation of the environmental contaminants in stem cell banks and can serve as initial evaluation for these establishments. The introduction of environmental monitoring programmes in the processing of stem cell lines could diminish the risk of contamination in stem cell cultures.     

土壤中有机污染物的老化概念探讨

有机污染物进入土壤后,随着其与土壤颗粒接触时间的延长,生物可利用性明显下降,发生老化现象,存在锁定行为.国内外已经广泛开展了与土壤中有机污染物老化相关的研究,但对老化的概念和内涵却没有明确界定,造成了理解上的偏差.基于目前国内外学者对老化的理解和认识,在明确生物可利用性的基础上,阐述了老化的概念及内涵,并对其进行了清晰的界定,同时对国内外现存的相关概念进行了区分.这些探讨将为相关研究的深入开展奠定基础,为土壤中有机污染物的环境化学行为与生物修复研究提供理论支持.     

Effects of heavy metals on immunocompetence of white-footed mice (Peromyscus leucopus)

Continued human population growth and industrialization result in increased contamination of wildlife habitats. Effects of such habitat deterioration on the well-being of natural populations are unclear. Exposure to contaminants may impair immunocompetence, thereby increasing disease susceptibility. The mammalian immune system is important in maintaining health and in its sensitivity to toxins. In our study conducted from May 1999 through May 2001, we examined assays of immnnoompetence in the white-footed mouse (Peromyscus leucopus) that inhabited reference sites and sites significantly contaminated with mixtures of heavy metals. We estimated potential exposure and uptake of heavy metals by measuring the level of each contaminant in representative soil and tissue samples. Intraindividual variation across mice, but not sex, explained a large portion of the overall variance in immune response, and spleen weight was significanltly afflicted by mouse age. We found no evidence that residence on contaminated sites had any effect on immunopathology and humoral immunity as measured in our study. We suggest that field and laboratory studies in ecotoxicology provide estimates of exposure to contaminants (i.e., tissue analyses) to establish a database suitable to clarify the dose-response relationship between contaminants and target systems.     

Phytoplankton bioassays for evaluating toxicity of in situ sediment contaminants

Routine bulk chemical characterization of sediments does not provide useful information on toxicity of sediment bound contaminants. This study reviewed and evaluated the utility of phytoplankton bioassays for evaluation of toxicity of sediment bound contaminants, including state-of-the-art techniques. Several techniques such as Algal Fractionation Bioassays, microcomputer-based toxicity testing and in situ bioassays including plankton cages have been developed and successfully applied in our research at various contaminated sites in the St. Lawrence Great Lakes. These bioassay techniques are sensitive, rapid and inexpensive for screening contaminants. The use and application of such techniques, based on bioavailability and physiological response of micro-organisms, are essential for the detection of environmental perturbations of an ecosystem. Such an early warning system will facilitate the preservation and rehabilitation of the Great Lakes.     

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.     

Monophyly of beta-tubulin and H+-ATPase gene variants in Glomus intraradices: consequences for molecular evolutionary studies of AM fungal genes

Arbuscular mycorrhizal fungi (AMF) are an ecologically important group of fungi. Previous studies showed the presence of divergent copies of beta-tubulin and V-type vacuolar H+-ATPase genes in AMF genomes and suggested horizontal gene transfer from host plants or mycoparasites to AMF. We sequenced these genes from DNA isolated from an in vitro cultured isolate of Glomus intraradices that was free of any obvious contaminants. We found two highly variable beta-tubulin sequences and variable H+-ATPase sequences. Despite this high variation, comparison of the sequences with those in gene banks supported a glomeromycotan origin of G. intraradices beta-tubulin and H+-ATPase sequences. Thus, our results are in sharp contrast with the previously reported polyphyletic origin of those genes. We present evidence that some highly divergent sequences of beta-tubulin and H+-ATPase deposited in the databases are likely to be contaminants. We therefore reject the prediction of horizontal transfer to AMF genomes. High differences in GC content between glomeromycotan sequences and sequences grouping in other lineages are shown and we suggest they can be used as an indicator to detect such contaminants. H+-ATPase phylogeny gave unexpected results and failed to resolve fungi as a natural group. beta-Tubulin phylogeny supported Glomeromeromycota as sister group of the Chytridiomycota. Contrasts between our results and trees previously generated using rDNA sequences are discussed.     

Contaminant Trends in Freshwater Fish from the Laurentian Great Lakes: A 20-Year Analysis

Although dietary concerns of Laurentian Great Lakes (GL) fish focus on the risk from persistent bioaccumulative toxicant (PBT) contaminants, fish are also an important source of nutrients beneficial to human health such as polyunsaturated fatty acids (e.g., eicosapentanoic acid and docosahexanoic acid). This study presents PBT trend data from the GL tribal fisheries over the past 20 years. PBT contaminants (282 analytes) from fillet portions of lake trout and whitefish were analyzed for trending patterns from 1992 to 2011 and are reported on five of the ATSDR/USEPA Great Lakes biomonitoring legacy contaminants (Hg, ΣDDE, ΣDDT, HCB, mirex, and ΣPCBs), two of the optional biomonitoring PBTs (toxaphene and Σdioxins/furans) and PCB 153 as a specific congener marker. Similar to other recent reports our data indicate that most PBT contaminant concentrations in the GL biota have decreased, which may indicate progress in reducing environmental emissions. Our research confirms that all contaminants demonstrate significant declines except Hg and toxaphene. Both of those remained constant after correcting for known independent factors of age, lipid, and size. These results are potentially encouraging and may provide useful data for the long distance and perhaps global influences of PBTs on the safety of fish consumption.     

Technical note: Examining the use of ethylenediaminetetraacetic acid for humic extraction of ancient bone

We examined the efficacy of ethylenediaminetetraacetic acid (EDTA) for removing humic contaminants from collagen extracted from ancient bone. Humic contaminants must be removed to obtain reliable stable isotope values from ancient bone collagen, given that humic acids have consistently lower δ¹³C values than collagen. The purpose of our research was to examine if EDTA treatment could effectively remove humic contaminants from bone collagen and thus serve as an alternative to the commonly implemented sodium hydroxide (NaOH) treatment, which may be associated with large collagen losses in poorly preserved samples. We compared the isotopic and elemental composition of ancient samples treated with EDTA alone, samples demineralized in hydrochloric acid (HCl) and rinsed in EDTA, samples treated with HCl alone, and samples demineralized in HCl and rinsed with NaOH. The samples used in the analyses were selected because they presented evidence of substantial humic contamination. We found that NaOH was the most effective agent for reducing humic contaminants as evidenced by the samples treated with this agent having higher δ¹³C values and lower C:N ratios relative to other treatments. The results from samples treated with EDTA suggest that this chemical cannot effectively remove humic contaminants given that these samples had significantly higher C:N ratios and lower δ¹³C and δ¹⁵N values relative to the HCl/NaOH treatment. Our results demonstrate that when performing stable isotope analysis of ancient bone collagen suspected to be contaminated with humic acids, NaOH is the most effective chemical agent to remove humic contaminants, while EDTA cannot perform this task.     

Modeling contaminants in AP-MS/MS experiments

Identification of protein-protein interactions (PPI) by affinity purification (AP) coupled with tandem mass spectrometry (AP-MS/MS) produces large data sets with high rates of false positives. This is in part because of contamination at the AP level (due to gel contamination, nonspecific binding to the TAP columns in the context of tandem affinity purification, insufficient purification, etc.). In this paper, we introduce a Bayesian approach to identify false-positive PPIs involving contaminants in AP-MS/MS experiments. Specifically, we propose a confidence assessment algorithm (called Decontaminator) that builds a model of contaminants using a small number of representative control experiments. It then uses this model to determine whether the Mascot score of a putative prey is significantly larger than what was observed in control experiments and assigns it a p-value and a false discovery rate. We show that our method identifies contaminants better than previously used approaches and results in a set of PPIs with a larger overlap with databases of known PPIs. Our approach will thus allow improved accuracy in PPI identification while reducing the number of control experiments required.     

Detection and drivers of exposure and effects of pharmaceuticals in higher vertebrates

Pharmaceuticals are highly bioactive compounds now known to be widespread environmental contaminants. However, research regarding exposure and possible effects in non-target higher vertebrate wildlife remains scarce. The fate and behaviour of most pharmaceuticals entering our environment via numerous pathways remain poorly characterized, and hence our conception and understanding of the risks posed to wild animals is equally constrained. The recent decimation of Asian vulture populations owing to a pharmaceutical (diclofenac) offers a notable example, because the exposure route (livestock carcasses) and the acute toxicity observed were completely unexpected. This case not only highlights the need for further research, but also the wider requirement for more considered and comprehensive ‘ecopharmacovigilance’. We discuss known and potential high risk sources and pathways in terrestrial and freshwater ecosystems where pharmaceutical exposure in higher vertebrate wildlife, principally birds and mammals, may occur. We examine whether approaches taken within existing surveillance schemes (that commonly target established classes of persistent or bioaccumulative contaminants) and the risk assessment approaches currently used for pesticides are relevant to pharmaceuticals, and we highlight where new approaches may be required to assess pharmaceutical-related risk.     

Phytoremediation—An Overview

The use of plants (directly or indirectly) to remediate contaminated soil or water is known as phytoremediation. This technology has emerged as a more cost effective, noninvasive, and publicly acceptable way to address the removal of environmental contaminants. Plants can be used to accumulate inorganic and organic contaminants, metabolize organic contaminants, and encourage microbial degradation of organic contaminants in the root zone. Widespread utilization of phytoremediation can be limited by the small habitat range or size of plants expressing remediation potential, and insufficient abilities of native plants to tolerate, detoxify, and accumulate contaminants. A better understanding and appreciation of the potential mechanisms for removing contaminants from the root zone and the interaction between plants, microorganisms, and contaminants will be useful in extending the application of phytoremediation to additional contaminated sites.     

Draft genome sequence of Sphingomonas echinoides ATCC 14820

Sphingomonas is a Gram-negative, yellow-pigmented, chemoheterotrophic, strictly aerobic bacterium. The bacterium is known to be metabolically versatile and can utilize a wide range of natural compounds as well as some types of environmental contaminants, such as creosote, polychlorinated biphenyls, etc. Here, we report the draft genome sequence of Sphingomonas echinoides ATCC 14820, which will provide additional information to enhance our understanding of metabolic versatility of Sphingomonas.     

Removal of chlorinated organic compounds during wastewater treatment: achievements and limits

A large number of chlorinated contaminants are found in wastewater, originating from domestic and industrial sources and from runoff captured by sewers. The presence of some of these contaminants, such as the pharmaceutical diclofenac, has recently been documented, whereas the presence of other contaminants, such as polychlorinated biphenyls, has been known for many years. This mini-review discusses the current state of knowledge regarding the degradation and outflow of chlorinated contaminants from municipal wastewater treatment plants (WWTPs) and the known physiologies of bacteria capable of degrading these contaminants, and summarizes current knowledge gaps as a way to focus future research efforts.     

Agranular membranes in free polysome preparations and their possible interference in studies of protein biosynthesis

1. Phospholipid-rich membranous contaminants are present in free polysomes from rat liver isolated on discontinuous sucrose gradients. 2. Electron-microscopic studies indicate that the membranous contaminants are mainly agranular with very occasional granular membranes. This is confirmed by the study of their sedimentation behaviour and their initial rate of labelling with radioactive glucosamine in vivo. 3. Conventional ribosome-purification methods fail to remove the contaminants, whereas deoxycholate effectively solubilizes the membranous contaminants with little breakdown of polysomes. 4. Amino acid-incorporation studies show that these membranous contaminants may seriously interfere in assessment of the functional capacity of free polysomes in protein biosynthesis in vivo.     

Ultraviolet radiation, toxic chemicals and amphibian population declines

Abstract. As part of an overall ‘biodiversity crisis’, many amphibian populations are in decline throughout the world. Numerous factors have contributed to these declines, including habitat destruction, pathogens, increasing ultraviolet (UV) radiation, introduced non‐native species and contaminants. In this paper we review the contribution of increasing UV radiation and environmental contamination to the global decline of amphibian populations. Both UV radiation and environmental contaminants can affect amphibians at all life stages. Exposure to UV radiation and to certain contaminants can kill amphibians and induce sublethal affects in embryos, larvae and adults. Moreover, UV radiation and contaminants may interact with one another synergistically. Synergistic interactions of UV radiation with contaminants can enhance the detrimental effects of the contaminant and UV radiation.     

Contaminants in biotechnologically manufactured l-tryptophan

The epidemic outbreak of a new disease, the eosinophilia-myalgia syndrome (EMS), was traced back to the intake of l-tryptophan (Trp) of certain lots from a single manufacturer. Since some trace contaminants were related to EMS, it appeared to be necessary to identify and find the origin of most trace contaminants in the EMS related Trp in order to apply this knowledge to a prospective manufacturing practice. Seventeen contaminants were determined in an implicated Trp lot by a single reversed-phase high-performance liquid chromatography run using UV and fluorescence detection. Most of these contaminants were classified as Trp metabolites, non-physiological oxidation or carbonyl condensation compounds of Trp. The amount and the pattern of contaminants were compared with recently manufactured Trp and Trp-containing preparations.     

Microbiological contamination in stem cell cultures

Cell therapy and regenerative medicine are potentially two of the most exciting aspects of the novel therapeutic methods currently under development. However, these treatments present a number of important biosafety issues, like the possible transmission of microorganisms to the recipients. The most common potential form of contamination in these cell products is by bacteria (including Mycoplasma), yeast and fungi. In our study, 32 stem cell lines and feeder cell lines were analysed. There were 19 contaminated cell passages (12%). The main contaminants were gram positive cocci and Mycoplasma species, followed by gram negative rods and gram positive rods. The Mycoplasma contamination rate was 4%. Stem cell banks and other research centres aim to screen all processed stem cell lines for these microorganisms, and to assure that no contaminants are introduced in the banking procedures. It is a standard part of current good practice in stem cell banks to carry out routine microbiological controls of the stem cell lines and to work in a controlled environment to reduce the probability of contamination in the final product.     

Chloramine T-mediated radioiodination of proteins on intact polyacrylamide slab gels

A simple, inexpensive method is described here for the radioiodination of proteins on intact, developed polyacrylamide gels. The method is based on the chloramine T iodination procedure which yields proteins containing ¹²⁵I label specifically in tyrosine residues. When employed with intact polyacrylamide gels, our method allows detection of proteins in amounts too small to be observed by chemical stains, including Coomassie blue. The procedure should, therefore, be useful for analyzing protein mixtures where only a small amount of material is available or for assaying trace contaminants in purified protein preparations. Proteins radioactively labeled by our method are suitable for further analysis by proteolytic cleavage followed by peptide mapping or “fingerprinting.”     

Interflow in semiarid environments: An overlooked process in risk assessment

Risk assessment, both human and ecological, embodies fundamental assumptions about hydrological processes, especially how they affect the movement of contaminants in the environment. The lateral movement of water through the soil, or interflow, is frequently a component of risk assessments for humid environments, but not of those for semiarid environments. Our research has shown that, contrary to what was previously thought, interflow can be important in semiarid landscapes and is, therefore, an essential consideration for risk assessment in these regions. To illustrate and assess the effect of interflow on estimates of risk, we (1) developed a simple conceptual model to describe the role that interflow may have in the redistribution of surface and near‐surface contamination, and (2) used RESRAD, an exposure model for assessing radionuclide doses to humans, to evaluate the effectiveness of landfill covers in mitigating doses of three contaminants (³H, ²³⁸U, and ²³⁹/²⁴⁰Pu) at a site in northern New Mexico at which interflow is known to be occurring. Only those calculations of the model that took interflow into account yielded the result that the radionuclides would contaminate groundwater‐underscoring the potential importance of interflow as a mechanism for the transport of contaminants. We conclude that failure to take interflow into account can render risk assessments inaccurate and remediation ineffective. Further, our work demonstrates that a general understanding of hydrological processes is essential for accurate risk assessment, ecological as well as human.