Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

One-dimensional and two-dimensional polyacrylamide gel electrophoresis: a tool for protein characterisation in aquatic samples

Dissolved organic nitrogen (DON) represents the least understood part of the nitrogen cycle. Due to recent methodological developments, proteins now represent a potentially characterisable fraction of DON at the macromolecular level. We have applied polyacrylamide gel electrophoresis to characterise proteins in samples from a range of aquatic environments in the Everglades National Park, Florida, USA. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that each sample has a complex and characteristic protein distribution. Some proteins appeared to be common to more than one site, and these might derive from dominant higher plant vegetation. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) provided better resolution; however, strong background hindered interpretation. Our results suggest that the two techniques can be used in parallel as a tool for protein characterisation: SDS-PAGE to provide a sample-specific fingerprint and 2D-PAGE to focus on the characterisation of individual protein molecules.

The assessment of TGGE for the detection of interspecific and intergeneric DNA-marker polymorphism within Solanaceae

RFLP markers are currently the most appropriate marker system for the identification of uncharacterised polymorphism at the interspecific and intergeneric level. Given the benefits of a PCR-based marker system and the availability of sequence information for many Solanaceous cDNA clones, it is now possible to target conserved fragments, for primer development, that flank sequences possessing interspecific polymorphism. The potential outcome is the development of a suite of markers that amplify widely in Solanaceae. Temperature gradient gel electrophoresis (TGGE) is a relatively inexpensive gel-based system that is suitable for the detection of most single-base changes. TGGE can be used to screen for both known and unknown polymorphisms, and has been assessed here, for the development of PCR-based markers that are useful for the detection of interspecific variation within Solanaceae. Fifteen markers are presented where differences between Lycopersicon esculentum and L. pennellii have been detected by TGGE. The markers were assessed on a wider selection of plant species and found to be potentially useful for the identification of interspecific and intergeneric polymorphism in Solanaceous plants.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Coleoptera and microbe biomass in Antarctic Dry Valley paleosols adjacent to the Inland Ice: Implications for Mars

Bulk paleosol samples collected from a Middle to Early Miocene moraine in the New Mountain area of the Dry Valleys, Antarctica, yielded Coleoptera exoskeletons and occasional endoskeletons showing considerable diagenetic effects along with several species of bacteria, all lodged in a dry-frozen but salt-rich horizon at shallow depth to the land surface. The till is at the older end of a chronologic sequence of glacial deposits, thought to have been deposited before the transition from wet-based to cold-based ice (similar to 15 Ma), and hence, entirely weathered in contact with the subaerial atmosphere. It is possible, though not absolutely verifiable, that the skeletons date from this early stage of emplacement having undergone modifications whenever light snowmelt occurred or salt concentrations lowered the freezing temperature to maintain water as liquid. Correlation of the Coleoptera species with cultured bacteria in the sample and the likelihood of co-habitation with Beauveria bassiani found in two adjacent, although younger paleosols, leads to new questions about the antiquity of the Coleoptera and the source of N and glucose from chitinase derived from the insects. The skeletons in the 831 section may date close to the oldest preserved chitin (Oligocene) yet found on Earth. While harsh Martian conditions make it seemingly intolerable for complex, multicellular organisms such as insects to exist in the near-surface and subaerially, life within similar cold, dry paleosol microenvironments (Cryosols) of Antarctica point to life potential for the Red Planet, especially when considering the relatively diverse microbe (bacteria and fungi) population. (C) 2011 Elsevier Ltd. All rights reserved.

Weathering rinds as mirror images of palaeosols: Examples from the Western Alps with correlation to Antarctica and Mars

Weathering rinds have been used for decades as relative age indicators to differentiate glacial deposits in long Quaternary sequences, but only recently has it been shown that rinds contain long and extensive palaeoenvironmental records that often extend far beyond mere repositories of chemical weathering on both Earth and Mars. When compared with associated palaeosols in deposits of the same age, rinds often carry a zonal weathering record that can be correlated with palaeosol horizon characteristics, with respect to both abiotic and biotic parameters. As demonstrated with examples from the French and Italian Alps, rinds in coarse clastic sediment contain weathering zones that correlate closely with horizon development in associated palaeosols of presumed Late Glacial age. In addition to weathering histories in both rinds and palaeosols, considerable evidence exists to indicate that the black mat impact (12.8 ka) reached the European Alps, a connection with the Younger Dryas readvance supported by both mineral and chemical composition. Preliminary metagenomic microbial analysis using density gradient gel electrophoresis suggests that the eubacterial microbial population found in at least one Ah palaeosol horizon associated with a rind impact site is different from that in other Late Glacial and Younger Dryas surface palaeosol horizons. © 2013 The Geological Society of London.

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

AIMS: The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota. METHODS AND RESULTS: A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples. CONCLUSIONS: Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. SIGNIFICANCE AND IMPACT OF STUDY: This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.

Estabilitat conformacional de variants de la ribonucleasa A: pressió i temperatura com a inductors dels desplegament proteic

A fi d'analitzar la contribució de la regió C-terminal proposada com a iniciadora del plegament (CFIS 106-118) a l'estabilitat de l'RNasa A, els residus alifàtics d'aquesta regió es van substituir, mitjançant mutagènesi dirigida, per altres residus en els quals la cadena lateral alifàtica era rogressivament escurçada. La major part de les substitucions projectades suposaven delecions no disruptives de grups metil(è). A més, es va reemplaçar la Tyr115 per un Trp, de manera que, potencialment, s'introduïa una única sonda fluorescent, no desestabilitzant, per tal de seguir els canvis conformacionals que es poguessin generar en la regió durant el procés de legament/desplegament de la proteïna. Tant els paràmetres cinètics, com els espectres d'FTIR i CD, determinats per cadascuna de les ribonucleases variants, indiquen que els reemplaçaments aminoacídics efectuats presenten, en general, poc o cap efecte en l'estructura nativa i en l'activitat de l'enzim. Es va emprar l'espectroscòpia d'absorció a l'ultraviolat de quarta derivada, la fluorescència (per la variant amb Trp) i l'espectroscòpia d'infraroig per transformada de Fourier, per tal de seguir i caracteritzar, en condicions d'equilibri, les transicions conformacionals de cada variant en funció de la pressió i de la temperatura. Els resultats es van comparar amb els que es van obtenir per la proteïna salvatge. Per determinar més a fons les característiques del procés de desplegament de la variant Y115W, les transicions de desnaturalització induïdes per urea d'aquesta variant i de la proteïna salvatge, van ésser examinades per mitjà d'electroforesi en gradient d'urea i espectroscòpia de fluorescència. Curiosament, els canvis conformacionals que resulten de la desnaturalització per pressió són molt semblants als que s'obtenen per temperatura. Enfront d'un augment gradual tant de pressió com de temperatura, l'estructura terciària i els elements d'estructura secundària de les proteïnes estudiades es perden de manera conjunta i reversible. Aquestes variacions estructurals que es promouen descriuen un procés de desplegament molt cooperatiu i en dos estats. Atès que ambdues tècniques (UV i FTIR) utilitzen cadascuna un règim de concentració proteica molt diferent, els resultats indiquen que el procés de desplegament per pressió i per temperatura és intramolecular. Els resultats obtinguts suggereixen que la hidrofobicitat i el volum de les cadenes laterals del CFIS, juntament amb les interaccions de van der Waals entre elements d'estructura secundària intervenen de manera molt notable en l'estabilització de la proteïna. Entre els diferents aminoàcids alifàtics que pertanyen al CFIS C-terminal, la Val108 és el residu més important per tal de preservar la integritat estructural de l'estat natiu. Els reemplaçaments en aquesta posició causen petites alteracions conformacionals i una gran desestabilització de la proteïna (per exemple, el punt mig de la transició de desnaturalització per pressió i per temperatura de la variant V108G disminueix uns 592 MPa i 25ºC, respectivament, respecte a la proteïna salvatge). D'acord amb els resultats obtinguts, la variant Y115W ofereix una sonda útil per tal de seguir la cinètica de plegament/desplegament de l'RNasa A.

Rifaximin modulates the colonic microbiota of patients with Crohn's disease: an in vitro approach using a continuous culture colonic model system.

Rifaximin, a rifamycin derivative, has been reported to induce clinical remission of active Crohn's disease (CD), a chronic inflammatory bowel disorder. In order to understand how rifaximin affects the colonic microbiota and its metabolism, an in vitro human colonic model system was used in this study. We investigated the impact of the administration of 1800 mg/day of rifaximin on the faecal microbiota of four patients affected by colonic active CD [Crohn's disease activity index (CDAI > 200)] using a continuous culture colonic model system. We studied the effect of rifaximin on the human gut microbiota using fluorescence in situ hybridization, quantitative PCR and PCR–denaturing gradient gel electrophoresis. Furthermore, we investigated the effect of the antibiotic on microbial metabolic profiles, using 1H-NMR and solid phase microextraction coupled with gas chromatography/mass spectrometry, and its potential genotoxicity and cytotoxicity, using Comet and growth curve assays. Rifaximin did not affect the overall composition of the gut microbiota, whereas it caused an increase in concentration of Bifidobacterium, Atopobium and Faecalibacterium prausnitzii. A shift in microbial metabolism was observed, as shown by increases in short-chain fatty acids, propanol, decanol, nonanone and aromatic organic compounds, and decreases in ethanol, methanol and glutamate. No genotoxicity or cytotoxicity was attributed to rifaximin, and conversely rifaximin was shown to have a chemopreventive role by protecting against hydrogen peroxide-induced DNA damage. We demonstrated that rifaximin, while not altering the overall structure of the human colonic microbiota, increased bifidobacteria and led to variation of metabolic profiles associated with potential beneficial effects on the host.

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

AIMS: The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota. METHODS AND RESULTS: A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples. CONCLUSIONS: Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. SIGNIFICANCE AND IMPACT OF STUDY: This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.

Investigation of the Europium Emission Spectra of the Europium-Oxytetracycline Complex in the Presence of Human Low-Density Lipoproteins

Low-Density Lipoprotein (LDL), often known as ""bad cholesterol"" is one of the responsible to increase the risk of coronary arterial diseases. For this reason, the cholesterol present in the LDL particle has become one of the main parameters to be quantified in routine clinical diagnosis. A number of tools are available to assess LDL particles and estimate the cholesterol concentration in the blood. The most common methods to quantify the LDL in the plasma are the density gradient ultracentrifugation and nuclear magnetic resonance (NMR). However, these techniques require special equipments and can take a long time to provide the results. In this paper, we report on the increase of the Europium emission in Europium-oxytetracycline complex aqueous solutions in the presence of LDL. This increase is proportional to the LDL concentration in the solution. This phenomenum can be used to develop a method to quantify the number of LDL particles in a sample. A comparison between the performances of the oxytetracycline and the tetracycline in the complexes is also made.

Epichlorohydrin Cross-Linking of Synthetic DNA Oligomers

Epichlorohydrin (ECH), an important chemical in the synthetic polymer industry, is a bifunctional alkylating agent with the potential to form DNA interstrand crosslinks. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown to undergo reaction with DNA in vivo and in vitro. We are using denaturing polyacrylamide gel electrophoresis to assess cross-linking of synthetic DNA oligomers by both ECH and the related compound, epibromohydrin (EBH). Both epihalohydrins produce a low-mobility band on denaturing gels consistent with an interstrand cross-link. Moreover, the efficiencies, sequence preferences, reaction kinetics, and pH dependence differ for the two compounds, suggesting different mechanisms of reaction. Understanding these alkylation reactions may help explain the role of the epihalohydrins in cancer development.

Esterase enzymes involved in pyrethroid and organophosphate resistance in a Brazilian population of Riphicephallus (Boophilus) microplus (Acari, Ixodidae)

Esterases are a group of enzymes that are reportedly associated with acaricide resistance in Riphicephallus (Boophilus) microplus. A comparative analysis was made of the esterase patterns in malathion and deltamethrin-sensitive, tolerant and resistant tick groups, using non-denaturing polyacrylamide gel electrophoresis. Electrophoretical profiles revealed four bands of esterase activity against alpha-naphthyl acetate; which were dubbed EST-1 to EST-4. The EST-3 and EST-4 were detected in all strains and were classified as carboxylesterases (CaEs). The EST-2, classified as an acetylcholinesterase (AChE), was detected in all groups, but its staining intensity increased from susceptible to resistant groups, indicating an altered production according to the degree of resistance. EST-1, which was also classified as an AChE, was detected exclusively in tolerant and resistant groups to both acaricides, but displayed greater activity in the malathion-resistant group. These data suggest that these AChEs may represent an important detoxification strategy developed to overcome the effects of acaricides. (C) 2008 Elsevier B.V. All rights reserved.

Spatial and Temporal Variation of Archaeal, Bacterial and Fungal Communities in Agricultural Soils

Background: Soil microbial communities are in constant change at many different temporal and spatial scales. However, the importance of these changes to the turnover of the soil microbial communities has been rarely studied simultaneously in space and time. Methodology/Principal Findings: In this study, we explored the temporal and spatial responses of soil bacterial, archaeal and fungal beta-diversities to abiotic parameters. Taking into account data from a 3-year sampling period, we analyzed the abundances and community structures of Archaea, Bacteria and Fungi along with key soil chemical parameters. We questioned how these abiotic variables influence the turnover of bacterial, archaeal and fungal communities and how they impact the long-term patterns of changes of the aforementioned soil communities. Interestingly, we found that the bacterial and fungal b-diversities are quite stable over time, whereas archaeal diversity showed significantly higher fluctuations. These fluctuations were reflected in temporal turnover caused by soil management through addition of N-fertilizers. Conclusions: Our study showed that management practices applied to agricultural soils might not significantly affect the bacterial and fungal communities, but cause slow and long-term changes in the abundance and structure of the archaeal community. Moreover, the results suggest that, to different extents, abiotic and biotic factors determine the community assembly of archaeal, bacterial and fungal communities.

EX-SITU BIOREMEDIATION OF BRAZILIAN SOIL CONTAMINATED WITH PLASTICIZERS PROCESS WASTES

The aim of this research was to evaluate the bioremediation of a soil contaminated with wastes from a plasticizers industry, located in Sao Paulo, Brazil. A 100-kg soil sample containing alcohols, adipates and phthalates was treated in an aerobic slurry-phase reactor using indigenous and acclimated microorganisms from the sludge of a wastewater treatment plant of the plasticizers industry (11gVSS kg(-1) dry soil), during 120 days. The soil pH and temperature were not corrected during bioremediation; soil humidity was corrected weekly to maintain 40%. The biodegradation of the pollutants followed first-order kinetics; the removal efficiencies were above 61% and, among the analyzed plasticizers, adipate was removed to below the detection limit. Biological molecular analysis during bioremediation revealed a significant change in the dominant populations initially present in the reactor.