Development and experimental validation of a nifH oligonucleotide microarray to study diazotrophic communities in a glacier forefield

Functional microarrays are powerful tools that allow the parallel detection of multiple strains at the species level and therefore to rapidly obtain information on microbial communities in the environment. However, the design of suitable probes is prone to uncertainties, as it is based so far on in silico predictions including weighted mismatch number and Gibbs free-energy values. This study describes the experimental selection of probes targeting subsequences of the nifH gene to study the community structure of diazotrophic populations present in Damma glacier (Swiss Central Alps) forefield soils. Using the Geniom^® One in situ synthesis technology (Febit, Germany), 2727 in silico designed candidate probes were tested. A total of 946 specific probes were selected and validated. This probe set covered a large diversity of the NifH phylotypes (35 out of the 45) found in the forefield. Hybridization predictors were tested statistically. Gibbs free-energy value for probe-target binding gave the best prediction for hybridization efficiency, while the weighted mismatch number was not significantly associated to probe specificity. In this study, we demonstrate that extensive experimental tests of probe-hybridization behaviour against sequences present in the studied environment remain a prerequisite for meaningful probe selection.     

Variations in soil culturable bacteria communities and biochemical characteristics in the Dongkemadi glacier forefield along a chronosequence

The variations in the soil culturable bacterial communities and biochemical parameters of early successional soils from a receding glacier in the Tanggula Mountain were investigated. We examined low organic carbon (C) and nitrogen (N) contents and enzymatic activity, correlated with fewer bacterial groups and numbers in the glacier forefield soils. The soil pH values decreased, but the soil water content, organic C and total N significantly increased, along the chronosequence. The soil C/N ratio decreased in the early development soils and increased in the late development soils and it did not correlate with the soil age since deglaciation. The activities of soil urease, sucrase, protease, polyphenol oxidase, catalase, and dehydrogenase increased along the chronosequence. The numbers of culturable bacteria in the soils increased as cultured at 25°C while decreased at 4°C from younger soils to older soils. Total numbers of culturable bacteria in the soils cultured at 25°C were significantly positively correlated to the soil total N, organic C, and soil water content, as well as the activities of soil urease, sucrase, dehydrogenase, catalase, and polyphenol oxidase. We have obtained 224 isolates from the glacier forefield soils. The isolates were clustered into 28 groups by amplified ribosomal DNA restriction analysis (ARDRA). Among them, 27 groups and 25 groups were obtained from the soils at 25°C and at 4°C incubation temperatures, respectively. These groups are affiliated with 18 genera that belong to six taxa, viz, Actinobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, Alphaproteobacteria, and Betaproteobacteria. The dominant taxa were Actinobacteria, Gammaproteobacteria, and Bacteroidetes in all the samples. The abundance and the diversity of the genera isolated at 25°C incubation temperature were greater than that at 4°C.     

Use of stochastic models to assess the effect of environmental factors on microbial growth

We present a novel application of a stochastic ecological model to the study and analysis of microbial growth dynamics as influenced by environmental conditions in an extensive experimental data set. The model proved to be useful in bridging the gap between theoretical ideas in ecology and an applied problem in microbiology. The data consisted of recorded growth curves of Escherichia coli grown in triplicate in a base medium with all 32 possible combinations of five supplements: glucose, NH(4)Cl, HCl, EDTA, and NaCl. The potential complexity of 2(5) experimental treatments and their effects was reduced to 2(2) as just the metal chelator EDTA, the presumed osmotic pressure imposed by NaCl, and the interaction between these two factors were enough to explain the variability seen in the data. The statistical analysis showed that the positive and negative effects of the five chemical supplements and their combinations were directly translated into an increase or decrease in time required to attain stationary phase and the population size at which the stationary phase started. The stochastic ecological model proved to be useful, as it effectively explained and summarized the uncertainty seen in the recorded growth curves. Our findings have broad implications for both basic and applied research and illustrate how stochastic mathematical modeling coupled with rigorous statistical methods can be of great assistance in understanding basic processes in microbial ecology.     

Application of a nifH oligonucleotide microarray for profiling diversity of N2-fixing microorganisms in marine microbial mats

Diazotrophic community structure in microbial mats from Guerrero Negro (GN), Baja California, Mexico, was studied using polymerase chain reaction amplification of the nifH gene and a newly developed nifH oligonucleotide microarray. Ninety-six oligonucleotide probes designed for nifH sequences from cultivated isolates and the environment were printed on glass microarrays. Phylogenetic analysis showed that the probes represented all of the main nifH clusters. Specificity was tested by (i) evaluation of cross hybridization using individual targets, and (ii) comparison of the observed hybridization signals and those predicted from the sequences cloned from microbial mats. Signal intensity had a positive relationship with target concentration and the percentage identity between probe and target. Under moderate stringency and high target concentration, specificity of the probes varied from 77% to 100% with the individual targets tested. At the end of a 7-month long nutrient manipulation experiment in GN microbial mats, no expression of nitrogen fixation under nitrogen loading was detected, although a diverse community of diazotrophs was detected. The diversity in diazotrophic population present was higher than in the population expressing the nifH gene, and there were taxa specific differences in response to nutrients. The nifH microarray is a powerful tool for diazotroph community analysis in the marine environment.     

Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenase enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNA gene tree to a considerable degree, which made it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterial communities in sediments of hyper saline and soda lakes. Although diazotrophs were revealed in all environmental samples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophic gammaproteobacteria were predominant in integrated samples. Analysis of the upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria, and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Validation of a portable EMG device to assess muscle activity during free-living situations

Portable amplifiers that record electromyograms (EMGs) for longer than four hours are commonly priced over $20,000 USD. This cost, and the technical challenges associated with recording EMGs during free-living situations, typically restrict EMG use to laboratory settings. A low-cost system (μEMG; OT Bioelecttronica, 100€), using specialized concentric bipolar electrodes, has been developed specifically for free-living situations. The purpose of this study was to validate the μEMG system by comparing EMGs from μEMG with a laboratory-based alternative (Telemyo 900; Noraxon USA, Inc.). Surface EMGs from biceps brachii (BB) and tibialis anterior (TA) of ten subjects were recorded simultaneously with both systems as subjects performed maximal voluntary contractions (MVCs), submaximal contractions at 25%, 50%, and 75% MVC, seven simulated activities of daily living (ADLs), and >60 min of simulated free-living inside the laboratory. In general, EMG parameters (e.g., average full-wave rectified EMG amplitude) derived from both systems were not significantly different for all outcome variables, except there were small differences across systems in baseline noise and absolute EMG amplitudes during MVCs. These results suggest that μEMG is a valid approach to the long-term recording of EMG.     

A method for calculating the dose absorbed by terrestrial animals to assess the environmental impact of radioactive contamination

A method for calculating the exposures of terrestrial animals in areas contaminated with radionuclides using a point source dose function is presented. To take into account scattered γ-radiation, the Berger formula for dose buildup factor in an infinite air medium has been parameterized. In the dosimetric model proposed, an animal phantom is presented as a parallelepiped to estimate external exposures and as a tissue-quivalent sphere to estimate internal doses. Using analytical expressions, dose rate conversion coefficients for external and internal exposures of animals have been estimated for individual radionuclides. For energies of γ-rays above 50 keV, the results are in good agreement with those estimated by the Monte Carlo method for ellipsoidal phantoms of animals.     

土壤微生物多样性及其环境影响因子研究进展

土壤中存在丰富的微生物资源,不同土壤系统具有不同的微生物群落,微生物多样性既依赖于生态系统又服务于生态系统。本文从物种多样性、遗传多样性、生态类型多样性、功能多样性四个方面对土壤微生物多样性进行了新的诠释,总结论述了土壤微生物多样性与环境因子如土壤、植物群落和气候条件之间的关系,并对目前存在的问题和今后面临的挑战提出几点看法。     

Correlation test to assess low-level processing of high-density oligonucleotide microarray data

Background  

There are currently a number of competing techniques for low-level processing of oligonucleotide array data. The choice of technique has a profound effect on subsequent statistical analyses, but there is no method to assess whether a particular technique is appropriate for a specific data set, without reference to external data.     

Design of a rotating disk reactor to assess the colonization of biofilms by free-living amoebae under high shear rates

The present study was aimed at designing and optimizing a rotating disk reactor simulating high hydrodynamic shear rates (γ), which are representative of cooling circuits. The characteristics of the hydrodynamic conditions in the reactor and the complex approach used to engineer it are described. A 60 l tank was filled with freshwater containing free-living amoebae (FLA) and bacteria. Adhesion of the bacteria and formation of a biofilm on the stainless steel coupons were observed. FLA were able to establish in these biofilms under γ as high as 85,000 s^?1. Several physical mechanisms (convection, diffusion, sedimentation) could explain the accumulation of amoeboid cells on surfaces, but further research is required to fully understand and model the fine mechanisms governing such transport under γ similar to those encountered in the industrial environment. This technological advance may enable research into these topics.     

Molecular method to assess the diversity of Burkholderia species in environmental samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.     

A review of the impact of environmental factors and pollutants on covid-19 transmission

Aerobiologia - The coronavirus disease (COVID-19) caused an unprecedented loss of life with colossal social and economic fallout over 237&nbsp;countries and territories worldwide. Environmental...     

A conceptual framework to assess the effects of environmental change on ecosystem services

A new conceptual framework is presented for the assessment of the impacts of environmental change drivers on ecosystem service provision and the policy and management responses that would derive from the valuation of these impacts. The Framework for Ecosystem Service Provision (FESP), is based on an interpretation of the widely-used Drivers-Pressures-State-Impact-Response (DPSIR) framework. FESP differs from the DPSIR by offering clarity in the definitions of the various DPSIR components as well as introducing novel elements of relevance to the ecosystem service approach. The value of a common framework lies in making the comparison across competing services accessible and clear as well as highlighting the conflicts and trade-offs between not only multiple ecosystem services, but also multiple service beneficiaries. The framework is explicit, for example, in recognising as state variables not only the attributes of the Ecosystem Service Providers (ESPs), but also the attributes of the Ecosystem Service Beneficiaries (ESBs). That a service depends as much on the attributes of the people whose well-being benefits from the service as on the attributes of the biology providing the service is an important step in integrated social-ecological thinking. FESP also identifies the mechanisms of either mitigation or adaptation to the environmental change problem through the effect of these response strategies on specific pressure or state variables. In this way, FESP can contribute to the policies and strategies that are used to support conservation management. This paper describes the principles of FESP and presents some indicative examples of its practical implementation.     

The impact of handling and environmental factors on the stress response and its consequences in swine

This review examines the effects of handling by humans and housing systems on physiological stress and some consequences in swine. Small amounts of threatening behavior by humans, imposed either regularly or irregularly, can produce a chronic stress response, as evidenced by increases in plasma free corticosteroid concentrations resulting in adverse effects on avoidance behavior, growth and reproductive performance. Housing systems involving a high level of confinement can result in a chronic stress response in swine with adverse effects on metabolism and the immune system. This stress response may be the result of unstable social relationships and can be ameliorated by the design of housing systems.     

Application of adenylate energy charge to problems of environmental impact assessment in aquatic organisms

Various physiological and biochemical methods have been proposed for assessing the effects of environmental perturbation on aquatic organisms. The success of these methods as diagnostic tools has, however, been limited. This paper proposes that adenylate energy charge overcomes some of these limitations. The adenylate energy charge (AEC) is calculated from concentrations of adenine nucleotides ([ATP+½ADP]/[ATP+ADP+AMP]), and is a reflection of metabolic potential available to an organism. Several features of this method are: correlation of specific values with physiological condition or growth state, a defined range of values, fast response times and high precision. Several examples from laboratory and field experiments are given to demonstrate these features. The test organisms used (mollusc species) were exposed to a variety of environmental perturbations, including salinity reduction, hydrocarbons and low doses of heavy metal. The studies performed indicate that the energy charge may be a useful measure in the assessment of environmental impact. Its use is restricted, however, as several limitations exist which need to be fully evaluated. Further work relating values to population characteristics of multicellular organisms needs to be completed before the method can become a predictive tool for management.     

Genome scan to assess the respective role of host-plant and environmental constraints on the adaptation of a widespread insect

Background  

The evolutionary success of phytophagous insects could result from their adaptation to different host-plants. Alternatively, the diversification of widespread species might be driven by adaptation along environmental gradients. To disentangle the respective roles of host-plant versus abiotic environmental variables acting on the genome of an oligophagous insect, we performed a genome scan using 83 unlinked AFLP markers on larvae of the large pine weevil collected on two host-plants (pine and spruce) in four forestry regions across Europe.     

Application of the microarray technique to cell blocks

OBJECTIVE: To evaluate whether the technique ofmicroarray construction can be applied to paraffin-embedded cell block specinmens. STUDY DESIGN: We used a manual microarray method for construction of a well-aligned cell microarray. First we evaluated the cellularity of the cell block and assigned the cases to the null, low, moderate or high cellularity category. Second, based on the different cellularity, we constructed 25 specimen cylinders into 1 block. We used routine hematoxylin-eosin (H-E) stain and immunocytochemistry (ICC) on the slide. RESULTS: All cell microarray paraffin block shaping and specimen cylinder arraying were finished in 1 step, so the specimen cylinders and the paraffin blocks of the cell microarray could very easily be incorporated. No sample was lost during the H-E staining process. However, a few samples fell off partially during the ICC process. Additionally, we observed that the higher cellularity groups yielded a better outcome as compared to the lower cellularity groups. CONCLUSION: This study introduced a very simple and economical technique for the creation of cell microarrays from cell blocks. This procedure should acquaint cytopathologists with microarray technology and encourage its use.