Atomic Force Microscopy Analysis of DNA Extracted from the Vegetative Cells and the Viable, but Nonculturable, Cells of Two Mycoplasmas ( Acholeplasma laidlawii

This article reports on a study of some characteristics of DNA extracted from the vegetative and viable, but nonculturable (VBNC), cells of two mycoplasma species (Acholeplasma laidlawii PG8 and Mycoplasma hominis PG37) using atomic force microscopy (AFM). DNA images were obtained by operating the AFM microscope in the tapping mode. It was found that DNA from the VBNC forms of M. hominis PG37 has decreased sizes (height: 0.177 ± 0.026 nm vs. 0.391 ± 0.041 nm for the vegetative forms, and width: 1.92 ± 0.099 vs. 2.17 ± 0.156 nm for the vegetative forms) in comparison to DNA from the vegetative forms of the mycoplasma. In the case of DNA from the A. laidlawii PG8 VBNC forms, we detected a decrease in width (1.506 ± 0.076 nm vs. 1.898 ± 0.117 nm for the vegetative forms), but an increase in height (0.641 ± 0.068 nm vs. 0.255 ± 0.010 nm for the vegetative forms) of the molecule. Analyzing the obtained results, one can speculate on some similarities in the physical-chemical properties of DNA from M. hominis PG37 and M. gallisepticum S6. In turn, this implies some general mechanisms of adaptation to a severe environment

Atomic Force Microscopy Analysis of DNA Extracted from the Vegetative Cells and the Viable, but Nonculturable, Cells of Two Mycoplasmas (Acholeplasma laidlawii PG8 and Mycoplasma hominis PG37)

This article reports on a study of some characteristics of DNA extracted from the vegetative and viable, but nonculturable (VBNC), cells of two mycoplasma species (Acholeplasma laidlawii PG8 and Mycoplasma hominis PG37) using atomic force microscopy (AFM). DNA images were obtained by operating the AFM microscope in the tapping mode. It was found that DNA from the VBNC forms of M. hominis PG37 has decreased sizes (height: 0.177 ± 0.026 nm vs. 0.391 ± 0.041 nm for the vegetative forms, and width: 1.92 ± 0.099 vs. 2.17 ± 0.156 nm for the vegetative forms) in comparison to DNA from the vegetative forms of the mycoplasma. In the case of DNA from the A. laidlawii PG8 VBNC forms, we detected a decrease in width (1.506 ± 0.076 nm vs. 1.898 ± 0.117 nm for the vegetative forms), but an increase in height (0.641 ± 0.068 nm vs. 0.255 ± 0.010 nm for the vegetative forms) of the molecule. Analyzing the obtained results, one can speculate on some similarities in the physical-chemical properties of DNA from M. hominis PG37 and M. gallisepticum S6. In turn, this implies some general mechanisms of adaptation to a severe environment. KEYWORDS: DNA, atomic force microscopy, mycoplasma, Acholeplasma laidlawii PG8, Mycoplasma hominis PG37 INTRODUCTION The inability of bacteria to grow on various bacteriological media, but saving their own metabolic activity at the same time, is referred to as the viable, but nonculturable (VBNC) state In the VBNC state, bacteria experience several morphological and physiological changes, including cell dwarfing (in particular, a shift from rod to coccoid form with decreased size) MATERIALS AND METHODS Acholeplasma laidlawii PG8 and M. hominis PG37 were obtained from the N. Cultivation of M. hominis PG37 for 3-5 days at 37°C was performed on the modified Edward's medium containing tryptose, 1.5% (w/v); HEPES, 0.2% (w/v); serum of horse blood, 10% (w/v); fresh yeast extract, 5% (w/v); arginine, 4% (w/v); benzylpenicillin (500,000 IE/ml), 0.2% (w/v); and phenol red, 0.3% (w/v). To obtain VBNC cells, glucose/arginine, serum of horse blood, yeast extracts, and phenol red were eliminated from Edward's medium; microorganisms were kept in this condition up to 13 months. To extract DNA from A. laidlawii PG8 and M. hominis PG37 cells, the following procedures were applied. Mycoplasma cultures (the vegetative and VBNC cells) were centrifuged at 9000g for 20 min. The pellet was resuspended in TES buffer (Tris-HCl 10 mM, EDTA 1 mM, NaCl 100 mM), SDS (1-2%) was added to the solution, then shaken and stored at 37°C for 5-10 min. An equal volume of aqueous phenol-chloroform mixture (1:1) was added to the obtained cell lysate. The aqueous phase was separated by centrifugation and the second extraction with chloroform-isoamyl alcohol mixture (24:1) was performed. DNA was precipitated with 2.5 volume of ethanol (96%). The pellet was dissolved in TE buffer (Tris-HCl 10 mM, Na-EDTA 1 mM, pH 8.0). For additional purification, the solution was incubated with RNAse (20 g/mL, Serva, Germany) for 30 min at 37°C and then with proteinase K (50 g/mL, Sigma, USA) for 30 min at 37°C. After the enzymatic treatment, DNA was again extracted with chloroform-isoamyl alcohol mixture and precipitated with 2.5 volume of ethanol (96%). DNA concentration was detected in agarose gel electrophoresis using standard DNA samples (Fermentas, Lithuania). The applied method allowed us to obtain DNA fragments that were then analyzed using AFM. A buffer containing 5 mM MgCl 2 and 10 mM Tris was used to dilute the obtained DNA samples to specific concentrations (0.15-0.5 ng/L). Since mica has a multilayer structure, it is necessary to eliminate the upper layer before coating samples. The upper layer of mica (Advanced Technologies Center, Moscow, Russia) was eliminated with tape. For this purpose, 1- 1-cm mica squares were placed onto the bottom of a 3-cm plastic Petri dish and covered superiorly with tape. When the tape was taken off, the upper layer of mica was eliminated. DNA samples (3 L) were placed onto the mica for 1 min. Trushin et al.: Investigation of Mycoplasma DNA TheScientificWorldJOURNAL (2010) 10, 894-900 896 Then the mica with DNA was rinsed twice with redistilled water and after each rinsing, it was dried with pressurized air. Images were acquired in air by a Solver P47H atomic force microscope (NT-MDT, Moscow, Russia) operated in the tapping mode using fpN11S cantilevers (r ≤ 10 nm, Advanced Technologies Center, Moscow, Russia). The height, Mag (signal from lock-in amplifier), RMS (signal from RMS detector), and phase (signal from the phase detector) were performed with the Nova 1.0.26 RC1 software (NT-MDT, Moscow, Russia). DNA height was measured directly using height regime in Nova 1.0.26 RC1. DNA width at the half-maximum was seen on the section of the DNA molecule with the compilation adjustment performed with the Nova 1.0.26 RC1 software. The Shapiro-Wilk test was applied to test whether our data yielded a normal distribution. Significant differences in the results were evaluated by applying Student's t test. A p value of <0.05 was considered significant. Data are given as mean SE. All calculations were made using the Origin 8.0 software for Windows. RESULTS AND DISCUSSION AFM is widely used to investigate DNA and DNA-protein complexes It is therefore reasonable to suggest that changes in cell physiology are consequences of alterations in the topology features of DNA. We previously reported that DNA of another mycoplasma, M. gallisepticum S6, had altered height and width values when the molecule was extracted from VBNC forms 897 the antiparallel single helices) are primary happenings in the chain of adaptation events. This may imply that similar DNA alterations for M. hominis PG37 and M. gallisepticum S6 may be responsible for the corresponding mechanisms of adjustment of the mycoplasmas to unfavorable growth conditions. Probably, A. laidlawii PG8 has a distinct mechanism of adaptation that reflects at the level of DNA by the different "molecule behavior". This suggestion should be tested in further experiments. ACKNOWLEDGMENT

Method for Identification of Threonine Isoforms in Peptides by Ultraviolet Photofragmentation of Cold Ions

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence

Microporous composite SiO2-TiO2 spheres prepared via the peroxo route: Lead(II) removal in aqueous media

Composite microporous SiO2-TiO2 spheres and micro/mesoporous TiO2 spheres were prepared via the template-free two-step synthetic route using aqueous peroxotitanate solution and tetraethyl orthosilicate (TEOS) as precursors. Both the composite SiO2-TiO2 and pure TiO2 spheres prepared by the solvent-exchange method were initially non-porous, but the applied reflux treatment in water-ethanol suspension successfully transformed them into microporous materials with high apparent surface areas approaching 500 m2·g− 1 and the micropore volume of 0.17 cm3·g− 1, while maintaining the same morphology. The prepared composites retained high values of pore volume and specific surface area up to 400 °C of thermal treatment temperature. The crystallization of TiO2 into the anatase phase in the mixed oxide occurred only at 700 °C, that process was also accompanied by the significant reduction of pore volume, as well as apparent surface area values. The prepared materials were tested as adsorbents for the lead(II) removal; they demonstrated high adsorption capacities, reaching 340 mg(Pb2 +)·g− 1. Moreover, the mixed silica-titania oxide was found to be more efficient adsorbent at low pH values.South Ural State University is grateful for financial support of the Ministry of Education and Science of the Russian Federation (grant No 16.2674.2014/K). University of Oviedo gratefully acknowledges financial support from the Spanish MINECO (MAT2013-40950-R and MAT2016-78155-C2-1-R). Igor Krivtsov thanks for the support the Russian Foundation for Basic Research 16-29-10757-ofi.Peer reviewe

Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses) in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing) to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity

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A new Fourier transform ion cyclotron resonance mass spectrometer based on a permanent magnet with an atmospheric pressure ionization source was designed and constructed. A mass resolving power (full-width-at-half-maximum) of up to 80,000 in the electron ionization mode and 25,000 in the electrospray mode was obtained. Also, a mass measurement accuracy at low-ppm level has been demonstrated for peptide mixtures in a mass range of up to 1200 m/z in the isotopically resolved mass spectra