On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

Genetically engineered binding proteins as biosensors for fermentation and cell culture

The signal-transduction properties and the potential applications of two engineered binding proteins from E. coli were extensively studied. Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP). Both proteins respond to their ligands in the micromolar range. The proteins can be stored at 4 degrees C for at least 5 months. Apparent binding constant, protein concentration, and fluorophore are three major factors that affect the biosensor's responsive ranges. The binding of the ligand is quick and reversible in solution, but the unfavorable dissociation equilibrium and mass-transfer resistance for encapsulated proteins can delay the response to several minutes and the recovery to hours. Simulated results show that using dialysis tubing with a diameter of 1 mm or less is possible to reduce the recovery time to less than 30 minutes. The potential applications of GBP were studied in yeast fermentation and E. coli fermentations in three different scales: 150 mL, 5 mL, and 100 microL. The results were compared with an YSI 2700 Chemistry Analyzer. Although the latter could not give reliable results for the E. coli fermentations as the glucose concentration in LB medium is close to its lower detection limit, the glucose biosensor presented here was successfully applied to each situation. Glutamine-binding protein was tested in cell cultures of two different scales (100 mL and 100 microL) and the results were also compared with those obtained with YSI. Both QBP and YSI gave good results for the 100-mL cell culture, but the relatively large sample volume requirement of YSI (at least 5 microL) prevented it from being used in the 100-microL cell culture. Because of their small sample volume requirements (less than 1 microL) and high sensitivity, the assays described here might find wide applications in high-throughput bioprocessing.     

Nanophotonic label-free biosensors for environmental monitoring

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Enzyme inhibition-based biosensors for food safety and environmental monitoring

Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different contaminants analyzed are considered. The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.     

Genetically engineered trees for plantation forests: key considerations for environmental risk assessment

Forests are vital to the world's ecological, social, cultural and economic well‐being yet sustainable provision of goods and services from forests is increasingly challenged by pressures such as growing demand for wood and other forest products, land conversion and degradation, and climate change. Intensively managed, highly productive forestry incorporating the most advanced methods for tree breeding, including the application of genetic engineering (GE), has tremendous potential for producing more wood on less land. However, the deployment of GE trees in plantation forests is a controversial topic and concerns have been particularly expressed about potential harms to the environment. This paper, prepared by an international group of experts in silviculture, forest tree breeding, forest biotechnology and environmental risk assessment (ERA) that met in April 2012, examines how the ERA paradigm used for GE crop plants may be applied to GE trees for use in plantation forests. It emphasizes the importance of differentiating between ERA for confined field trials of GE trees, and ERA for unconfined or commercial‐scale releases. In the case of the latter, particular attention is paid to characteristics of forest trees that distinguish them from shorter‐lived plant species, the temporal and spatial scale of forests, and the biodiversity of the plantation forest as a receiving environment.     

Bio-indicators in the monitoring of environmental pollution

Summary Surveying the chemical pollution status of a given area is increasingly becoming the task of biological indicators, ie animal and plant organisms capable of providing us with the necessary data. For example, in the wake of certain contaminations, they may increase or decrease in number, vary population proportions in relation to given traits, become vectors of certain toxic molecules or heavy metals, or accumulate them in their bodies or in their «products». The advantage of bioindicators over chemical or physical detectors is their ability to supply extensive — both spatially and temporally —rather than limited and instantaneous data, thus making such information more representative. In many instances the bioindicator takes samples for us-a service that is undoubtedly valuable even though it must be linked to a sound knowledge of the organism's ethogram and biology so as to arrive at a scientifically legitimate interpretation of the data provided.     

Genetically engineered livestock for biomedical models

To commemorate Transgenic Animal Research Conference X, this review summarizes the recent progress in developing genetically engineered livestock species as biomedical models. The first of these conferences was held in 1997, which turned out to be a watershed year for the field, with two significant events occurring. One was the publication of the first transgenic livestock animal disease model, a pig with retinitis pigmentosa. Before that, the use of livestock species in biomedical research had been limited to wild-type animals or disease models that had been induced or were naturally occurring. The second event was the report of Dolly, a cloned sheep produced by somatic cell nuclear transfer. Cloning subsequently became an essential part of the process for most of the models developed in the last 18 years and is stilled used prominently today. This review is intended to highlight the biomedical modeling achievements that followed those key events, many of which were first reported at one of the previous nine Transgenic Animal Research Conferences. Also discussed are the practical challenges of utilizing livestock disease models now that the technical hurdles of model development have been largely overcome.     

Molecular methods for environmental monitoring and containment of genetically engineered microorganisms

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.     

In situ microbial metabolism of aromatic-hydrocarbon environmental pollutants

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Highlights► Microorganisms play a crucial role in detoxifying environmental pollutants. ► Proving that biodegradation processes occur in the real world (in situ) is important. ► Genetics, biochemistry, and physiology advance the science of microbial metabolism. ► Such advances provide new molecular-biomarker tools for documenting biodegradation. ► Applying new tools to contaminated sites reveals new microbial processes and diversity.     

Genetically engineered pig models for diabetes research

Diabetes mellitus (DM) has emerged into a steadily increasing health problem and the predicted future dimension of the global DM epidemic is alarming: an increase from currently 346 million to over 400 million affected people worldwide by the year 2030 was extrapolated. Thus concerted research efforts are imperative to gain insight into disease mechanisms and to expand the basis for development of preventive and therapeutic strategies. Diabetic rodent models have traditionally been used to follow these goals, but have limitations for translational research. The pig is another classical animal model for diabetes research. Genetic engineering now facilitates tailoring pig models which mimic human disease mechanisms at the molecular level. This article reviews the existing genetically engineered pig models for diabetes research and their current and future applications. Further, the potential role of the pig as donor of pancreatic islets for xenotransplantation or as host for growing human pancreas is outlined.     

新型吡唑类化合物DL-1的硝化抑制效应初探

以国内外应用较为广泛的硝化抑制剂双氰胺（DCD）为参比对象,采用室内培养方法,对新型吡唑类化合物DL-1的硝化抑制效应进行初步探讨.结果表明,DL-1对土壤中铵的氧化过程具有显著的抑制效应,前3周的硝化抑制率可达70％以上,且硝化抑制能力在第14天至28天最强.与等量DCD相比,施用量为(NH₄)₂SO₄氮量1.0%的DL-1在14、21和28 d使土壤中的NO₃^--N含量分别下降 26.23%、33.27%和23.31%;与不加抑制剂的对照处理相比,土壤NO₃^--N含量则分别下降了71.12%、69.10%和55.14%.当DL-1用量为(NH₄)₂SO₄氮量的2。0%时,土壤的硝化作用受到了更强烈的抑制,到培养第90天试验结束,土壤中的NO₃^--N含量始终维持在较低水平.     

Phylogenetic and functional metagenomic profiling for assessing microbial biodiversity in environmental monitoring

Decisions guiding environmental management need to be based on a broad and comprehensive understanding of the biodiversity and functional capability within ecosystems. Microbes are of particular importance since they drive biogeochemical cycles, being both producers and decomposers. Their quick and direct responses to changes in environmental conditions modulate the ecosystem accordingly, thus providing a sensitive readout. Here we have used direct sequencing of total DNA from water samples to compare the microbial communities of two distinct coastal regions exposed to different anthropogenic pressures: the highly polluted Port of Genoa and the protected area of Montecristo Island in the Mediterranean Sea. Analysis of the metagenomes revealed significant differences in both microbial diversity and abundance between the two areas, reflecting their distinct ecological habitats and anthropogenic stress conditions. Our results indicate that the combination of next generation sequencing (NGS) technologies and bioinformatics tools presents a new approach to monitor the diversity and the ecological status of aquatic ecosystems. Integration of metagenomics into environmental monitoring campaigns should enable the impact of the anthropogenic pressure on microbial biodiversity in various ecosystems to be better assessed and also predicted.     

Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos

The design of a biosensor for the detection of dichlorvos at attomolar levels is described based on a highly sensitive double mutant (E69Y Y71D) of the Drosophila melanogaster acetylcholinesterase (Dm. AChE). This enzyme has a k(i) for dichlorvos equal to 487 microM(-1)min(-1), which is 300 and 20,000 times higher than that of the wild type Dm. AChE and the Electrophorus electricus AChE (E.el. AChE), respectively. The enzyme is immobilized into microporous-activated conductive carbon, and is used as such for the development of an inhibitor electrochemical biosensor. This E69Y Y71D mutant enables the decrease in the detection limit of the biosensor down to 10(-17) M, which is five orders of magnitude lower compared to the Electropharus electricus-based biosensor and eight orders of magnitude lower than the biosensors described so far.     

A defined substrate technology for the enumeration of microbial indicators of environmental pollution

The examination of water and other environmental sources for microbial pollution is a major public health undertaking. Currently, there are two accepted methods in use: the multiple-tube fermentation (MTF) and the membrane filtration (MF) tests. Both methods are designed to enumerate the secondary indicator group, total coliforms. Both tests suffer several inherent limitations, including a time delay of three to seven days to obtain a definitive result, the subjective nature of the test interpretation, and the inability to provide directly useful public health information. A defined substrate technology, originally used to enumerate specific bacterial species from mixtures in clinical urine specimens, was applied to water testing; the technology was constituted to enumerate simultaneously both total coliforms and the primary indicator bacterium E. coli. Examination of environmental isolates of these two classes of target microbes showed sensitivity equal to available methods, with potentially greater specificity. It was not subject to inhibition by bacteria other than the targets, grew injured coliforms, did not require confirmatory tests, and the maximum time to a positive was 24 hours. The defined substrate technology provides both regulatory and directly useful public health information.     

Activation analysis of human hair as a tool for environmental pollution monitoring

Recent development and uses of neutron activation techniques for human hair analyses are reviewed. The method of neutron activation analysis (NAA) appears to have the potential to be used as a tool for environmental pollution monitoring. Principally, two types of NAA procedure are in use nowadays for multielement analyses of human scalp hair. The more common of these is the method of instrumental neutron activation analysis (INAA), consisting of a single short-term (3-10 hours) exposure of hair to a beam of neutrons in a nuclear reactor, followed by two measurements of gamma-ray spectra at 2-3 days and 3-4 weeks after the end of irradiation. The following microelements can be commonly determined by this type of activation procedure: As, Au, Br, Cu, K, La, Na, Sb, Sm, Co, Cr, Cs, Fe, Hg, Rb, Sc, Se and Zn. The other of the two procedures involves the use of radiochemical separation techniques and is employed for quantitative determinations of elements that are not easily determined by INAA (Mo, Cd, Ni, etc.), or in cases where there is a need to achieve the lowest possible limits of analytical determination. The accuracy of NAA techniques is strongly dependent on the hair sampling and hair sample processing methods used. The analytical error of this method may vary within the range of 5-15%. Its applicability as a tool for monitoring the environmental pollution level is here demonstrated on an example of groups of individuals living in the areas differing by the degree of environmental pollution. The use of other biopsy materials, such as e.g. mammalian hair, for the purpose of environmental exposure monitoring is also considered in this review.