Green approach for ultratrace determination of divalent metal ions and arsenic species using total-reflection X-ray fluorescence spectrometry and mercapto-modified graphene oxide nanosheets as a novel adsorbent.

A new method based on dispersive microsolid phase extraction (DMSPE) and total-reflection X-ray fluorescence spectrometry (TXRF) is proposed for multielemental ultratrace determination of heavy metal ions and arsenic species. In the developed methodology, the crucial issue is a novel adsorbent synthesized by grafting 3-mercaptopropyl trimethoxysilane on a graphene oxide (GO) surface. Mercapto-modified graphene oxide (GO-SH) can be applied in quantitative adsorption of cobalt, nickel, copper, cadmium, and lead ions. Moreover, GO-SH demonstrates selectivity toward arsenite in the presence of arsenate. Due to such features of GO-SH nanosheets as wrinkled structure and excellent dispersibility in water, GO-SH seems to be ideal for fast and simple preconcentration and determination of heavy metal ions using methodology based on DMSPE and TXRF measurement. The suspension of GO-SH was injected into an analyzed water sample; after filtration, the GO-SH nanosheets with adsorbed metal ions were redispersed in a small volume of internal standard solution and deposited onto a quartz reflector. The high enrichment factor of 150 allows obtaining detection limits of 0.11, 0.078, 0.079, 0.064, 0.054, and 0.083 ng mL(-1) for Co(II), Ni(II), Cu(II), As(III), Cd(II), and Pb(II), respectively. Such low detection limits can be obtained using a benchtop TXRF system without cooling media and gas consumption. The method is suitable for the analysis of water, including high salinity samples difficult to analyze using other spectroscopy techniques. Moreover, GO-SH can be applied to the arsenic speciation due to its selectivity toward arsenite.

Evaluation of the antibacterial activity of cystatin against selected strains of Escherichia coli.

The aim of this study was to analyze the antibacterial activity of hen egg white cystatin against selected Escherichia coli strains. We used a monomeric solution of hen egg white cystatin in bovine serum albumin (BSA) with added phosphate buffered saline (PBS), and three test strains: Escherichia coli ATCC 23811, Escherichia coli ATCC 8739 and Escherichia coli ATCC 25922. The effect of cystatin against the tested strains was determined on the basis of minimal inhibitory concentration (MIC) and survival curves of the microorganisms in a cystatin-containing environment during incubation at various temperatures. Our study confirmed the activity of cystatin against the analyzed Escherichia coli strains. taining environment, as compared to control samples incubated in a ovocystatin-deficient medium. Depending on the incubation temperature (20 degrees C or 37 degrees C) the reduction persisted up to 12 hours after incubation.

Importance of type specimen study for understanding genus boundaries-taxonomic clarifications in Lepidoderma based on integrative taxonomy approach leading to resurrection of the old genus Polyschismium.

Type specimens of four species of Lepidoderma (Myxomycetes, Amoebozoa)-L. crassipes, L. neoperforatum, L. perforatum, and L. stipitatum-have been studied using an integrative approach including application of traditional taxonomy methods, i.e., morphological study under stereoscopic and compound microscopes, detailed analysis of micromorphological characters using scanning electron microscopy, and molecular analysis by way of Sanger sequencing of molecular markers (nuc 18S rDNA and elongation factor 1-alpha gene, EF1A). Results of the study revealed that L. crassipes is conspecific with L. tigrinum, L. stipitatum is a malformed specimen of Diderma floriforme, whereas L. perforatum and L. neoperforatum represent two well-defined morphologically and genetically separate species. Phylogeny of Physarales shows the polyphyletic character of the genus Lepidoderma. The type species of Lepidoderma clusters together with Diderma, whereas other representatives of this genus form a monophyletic, well-supported clade. The species from this clade are proposed to belong to the genus Polyschismium described by A. Corda in 1842 that is resurrected and emended here. Nine species of Lepidoderma are transferred to Polyschismium. A new key to Didymiaceae including Polyschismium is provided.

Didymium pseudonivicola: A new myxomycete from the austral Andes emerges from broad-scale morphological and molecular analyses of D. nivicola collections.

A new nivicolous myxomycete is described as a result of a comprehensive study of Didymium nivicola collections from the entire range of its occurrence. Statistical analysis of 12 morphological characters, phylogenetic analyses of nuc 18S rDNA and elongation factor 1-alpha gene (EF1A), and a delimitation method (automatic barcode gap diversity) have been applied to corroborate the identity of the new species. A preliminary morphological analysis of D. nivicola revealed high variability of South American populations where four types of spore ornamentation were noted. However, results of molecular study and statistical analysis of morphological characters did not support recognition of these four forms but the distinction of two morphotypes. Consequently, two species have been recognized: D. nivicola and the newly proposed D. pseudonivicola. The new species can be distinguished from D. nivicola by distinctly larger and mostly plasmodiocarpic sporophores, which are scattered to gregarious, paler spores, and by the paler, more delicate and more elastic capillitium. Spore ornamentation of D. pseudonivicola is uniform and can be described as distinctly spiny (pilate under scanning electron microscope [SEM]), whereas those of D. nivicola is more variable, where spines (pilae under SEM) are delicate, distinct, or conspicuous. Additionally, whereas D. nivicola is a species distributed worldwide, D. pseudonivicola occurs only in the austral Andes of Argentina and Chile.

Range-wide Phylogeography of a Nivicolous Protist Didymium nivicola Meyl. (Myxomycetes, Amoebozoa): Striking Contrasts Between the Northern and the Southern Hemisphere.

Soil protists play a crucial role in terrestrial ecosystems and often show immense taxonomic diversity. However, for many groups, distribution patterns remain largely unknown. We investigated range-wide intraspecific diversity of a specialized airborne protist (Didymium nivicola Meyl.) that occupies a narrow ecological niche associated with long-lasting snow cover. We sampled 122 collections covering all areas where the species was recorded worldwide. We obtained 105 and 41 sequences of small ribosomal subunit rDNA (SSU) and elongation factor 1-alpha (EF1A), respectively. While the species is very diverse in the austral Andes, Southern Hemisphere (SH; 17 SSU ribotypes and 12 EF1A genotypes identified), its populations are genetically uniform across three continents of the Northern Hemisphere (NH; single ribotype, single genotype). Our results indicate the austral Andes as a possible diversification centre for D. nivicola where populations seem to reproduce sexually. Two main parts of the range display highly contrasting genetic patterns, thus biogeographical history and dynamics. Current distribution of D. nivicola in the NH is likely a result of a dispersal event from the SH and subsequent long-distance dispersal (LDD) that might be associated with a shift to asexual mode of reproduction.

New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes.

Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.

Nivicolous Trichiales from the austral Andes: unexpected diversity including two new species.

Nivicolous myxomycetes are a group of amoebozoan protists dependent on long-lasting snow cover worldwide. Recent fine-scale analysis of species diversity from the austral Andes revealed high intraspecific variability of most taxa, suggesting independent evolutionary processes and significant differences in species compositions between the Northern (NH) and Southern (SH) Hemispheres. The present study is the second part of this analysis based on representatives of Trichiales. A total of 173 South American collections were studied based on morphological and molecular data, and 15 taxa have been identified. Two of them, Hemitrichia crassifila and Perichaena patagonica, are proposed as new species confirmed by a phylogeny of Trichiales. However, their affinity to the genera in which they are proposed are not confirmed due to polyphyletic character of all genera of Trichiales. Four species, Dianema subretisporum, Trichia contorta var. karstenii, T. nivicola, and T. sordida, are reported for the first time from the Southern Hemisphere. One species, T. alpina, is new for Argentina. Additionally, we provide the first record of Perichaena megaspora from Chile. Specimen frequency and species diversity of Trichiales found at nivicolous localities in the austral Andes are unexpectedly high, exceeding those of Stemonitidales, the most numerous group in the Northern Hemisphere, where Trichiales play a marginal role. By contrast, Trichiales appear the main component of nivicolous assemblages in the Andes. Results of the present work, together with the earlier analysis of Stemonitidales, indicate that the Andes constitute an exceptionally important evolutionary hot spot for nivicolous myxomycetes characterized by an outstanding species diversity.

Selective adsorption and determination of hexavalent chromium ions using graphene oxide modified with amino silanes.

Novel adsorbents are described for the preconcentration of chromium(VI). Graphene oxide (GO) was modified with various amino silanes containing one, two, or three nitrogen atoms in the molecule. These include 3-aminopropyltriethoxysilane (APTES), N-(3-trimethoxysilylpropyl)ethylenediamine (TMSPEDA), and N1-(3-trimethoxysilylpropyl)diethylenetriamine (TMSPDETA). The resulting GO derivatives were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and energy-dispersive X-ray fluorescence spectrometry (EDXRF). Adsorption studies show that these GO based sorbents are highly selective for Cr(VI) in the presence of Cr(III) at pH 3.5. Although the amino silanes applied in modification of GO contain different numbers of nitrogen atoms, the maximum adsorption capacities of GO derivatives are very similar (13.3-15.1 mg·g-1). Such results are in accordance with spectroscopy studies which show that the amount of amino silanes attached to GO decreases in the order of APTES > TMSPEDA > TMSPDETA. The APTES-modified GO was applied to selective and sensitive extraction of Cr(VI) ions prior to quantitation by low-power EDXRF using the Cr Kα line. The Cr(VI) ions need not be eluted from the solid adsorbent. The method has a 0.17 ng·mL-1 detection limit, and the recovery is 99.7 ± 2.2% at a spiking level of 10 ng·mL-1. The method was successfully applied to the determination of Cr(VI) in water samples. Graphical abstractGraphene oxide adsorbents modified with various amino silanes are described for the preconcentration and speciation of trace and ultratrace levels of chromium ions.

What an Intron May Tell: Several Sexual Biospecies Coexist in Meriderma spp. (Myxomycetes).

Specimens of the snowbank myxomycete Meriderma atrosporum agg. from five European mountain ranges were sequenced for parts of the nuclear small subunit ribosomal RNA gene (SSU) and the protein elongation factor 1 alpha gene (EF1A). A phylogeny of the EF1A gene, including a very variable spliceosomal intron, resulted in seven phylogroups, and this topology was confirmed by SSU sequences. Two thirds of all specimens were heterozygous for the EF1A gene, and the two haplotypes of these specimens occurred always in the same phylogroup. Except for two cases in closely related phylogroups all ribotypes were as well limited to one phylogroup. This pattern is consistent with the assumption of reproductively isolated sexual biospecies. Numbers of EF1A-haplotypes shared between mountain ranges correlate with geographical distance, suggesting relative isolation but occasional long-distance dispersal by spores. Most subpopulations (divided by putative biospecies and mountain ranges) were in Hardy-Weinberg equilibrium. A simulation assuming panmixis within but not in between subpopulations suggested that similar numbers of shared genotypes can be created by chance through sexual reproduction alone. Our results support the biospecies concept, derived from experiments with cultivable members of the Physarales. We discuss the results on the background of possible reproductive options in myxomycetes.

Suspended aminosilanized graphene oxide nanosheets for selective preconcentration of lead ions and ultrasensitive determination by electrothermal atomic absorption spectrometry.

The aminosilanized graphene oxide (GO-NH2) was prepared for selective adsorption of Pb(II) ions. Graphene oxide (GO) and GO-NH2 prepared through the amino-silanization of GO with 3-aminopropyltriethoxysilane were characterized by scanning electron microscopy, powder X-ray diffraction, X-ray photoelectron spectroscopy, and Raman spectroscopy. The batch experiments show that GO-NH2 is characterized by high selectivity toward Pb(II) ions. Adsorption isotherms suggest that sorption of Pb(II) on GO-NH2 nanosheets is monolayer coverage, and adsorption is controlled by a chemical process involving the surface complexation of Pb(II) ions with the nitrogen-containing groups on the surface of GO-NH2. Pb(II) ions can be quantitatively adsorbed at pH 6 with maximum adsorption capacity of 96 mg g(-1). The GO-NH2 was used for selective and sensitive determination of Pb(II) ions by electrothermal atomic absorption spectrometry (ET-AAS). The preconcentration of Pb(II) ions is based on dispersive micro solid-phase extraction in which the suspended GO-NH2 is rapidly injected into analyzed water sample. Such features of GO-NH2 nanosheets as wrinkled structure, softness, flexibility, and excellent dispersibility in water allow achieving very good contact with analyzed solution, and adsorption of Pb(II) ions is very fast. The experiment shows that after separation of the solid phase, the suspension of GO-NH2 with adsorbed Pb(II) ions can be directly injected into the graphite tube and analyzed by ET-AAS. The GO-NH2 is characterized by high selectivity toward Pb(II) ions and can be successfully used for analysis of various water samples with excellent enrichment factors of 100 and detection limits of 9.4 ng L(-1).

Spherical silica particles decorated with graphene oxide nanosheets as a new sorbent in inorganic trace analysis.

Graphene oxide (GO) is a novel material with excellent adsorptive properties. However, the very small particles of GO can cause serious problems is solid-phase extraction (SPE) such as the high pressure in SPE system and the adsorbent loss through pores of frit. These problems can be overcome by covalently binding GO nanosheets to a support. In this paper, GO was covalently bonded to spherical silica by coupling the amino groups of spherical aminosilica and the carboxyl groups of GO (GO@SiO2). The successful immobilization of GO nanosheets on the aminosilica was confirmed by scanning electron microscopy and X-ray photoelectron spectroscopy. The spherical particle covered by GO with crumpled silk wave-like carbon sheets are an ideal sorbent for SPE of metal ions. The wrinkled structure of the coating results in large surface area and a high extractive capacity. The adsorption bath experiment shows that Cu(II) and Pb(II) can be quantitatively adsorbed at pH 5.5 with maximum adsorption capacity of 6.0 and 13.6 mg g(-1), respectively. Such features of GO nanosheets as softness and flexibility allow achieving excellent contact with analyzed solution in flow-rate conditions. In consequence, the metal ions can be quantitatively preconcentrated from high volume of aqueous samples with excellent flow-rate. SPE column is very stable and several adsorption-elution cycles can be performed without any loss of adsorptive properties. The GO@SiO2 was used for analysis of various water samples by flame atomic absorption spectrometry with excellent enrichment factors (200-250) and detection limits (0.084 and 0.27 ng mL(-1) for Cu(II) and Pb(II), respectively).

Elemental composition of Physarum compressum Alb. et Schw. sporocarps and their structures cultivated on rabbit dung and agar substrates.

The elemental composition of spores, peridium walls, and lime nodes of Physarum compressum sporocarps, cultivated on rabbit dung as a natural growing environment for the slime mold and on artificial agar medium, was compared to evaluate differences that may be dependent on substrates. Whole fruiting bodies and samples of both experimental media were extracted with nitric acid or Parr digest bomb, respectively, and analyzed by means of total X-ray reflection fluorescence (TXRF). Electron probe microanalysis (EPMA) of spores, peridium walls, and lime nodes structure was carried out with the scanning electron microscope equipped with energy-dispersive spectrometer. Because of minute sizes and roughness of investigated structures, Monte Carlo simulations were utilized to establish analytical conditions of EPMA. Biological and geological standards were used in the quantification of element concentrations. According to TXRF, the fruiting bodies from agar medium revealed lower concentrations of K, Ca, Cr, Mn, and Fe in relation to fruiting bodies from the dung, reflecting elemental relationships in the experimental media. According to EPMA, the highest Ca concentration was found in the lime nodes followed by the peridium and the spores. Culturing of the slime molds on the rabbit dung indicated higher concentration of Ca in the lime nodes and peridium walls when compared with those obtained from the sporocarps grown on agar media. The opposite relation was found for the spores. The concentration of Na, Mg, P, S, and Cl was generally lower in all structures of the sporocarps harvested from the dung than from the agar medium. K was in higher concentration in analyzed structures from dung than from agar. Different element uptake (except for Ca and K) was revealed by the two methods: TXRF and EPMA.