Benchmarking DNA isolation methods for marine metagenomics.

Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a "kitome", e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.

Examining the applicability of polar organic chemical integrative sampler for long-term monitoring of groundwater contamination caused by currently used pesticides.

The possibilities of expanding a groundwater quality monitoring scheme by passive sampling using polar organic chemical integrative sampler (POCIS) comprising HLB sorbent as the receiving phase were explored. Passive sampling and grab sampling were carried out simultaneously in the regions with vulnerable groundwater resources in Slovakia, between 2013 and 2021. For 27 pesticides and degradation products detected both in POCIS and the grab samples, in situ sampling rates were calculated and statistically evaluated. The limited effectiveness of the receiving phase in POCIS for sampling polar or ionized compounds was confirmed through a comparison of the medians of compound-specific sampling rates. For the majority of the monitored compounds the median sampling rates varied between 0.01 and 0.035 L/day. In some cases, the actual in situ values could be confirmed by parallel exposure of POCIS and silicone rubber sheet employed to obtain a benchmark for maximum attainable sampling rate. Sampling site and sampling period appear to have also some influence on the sampling rates, which was attributed in part to the groundwater velocity varying in both space and time. The influence of physico-chemical parameters (temperature, pH, electrolytic conductivity) remains mostly questionable due to the naturally limited ranges of recorded values over the entire duration of the study. Concentrations of pollutants in POCIS could be used for predicting time weighed average concentrations in water, provided the sampling rates were known and relatively constant. Generally, the compound-specific sampling rate cannot be considered constant due to a combination of naturally varying environmental factors that influence the actual in situ sampling rate. The relative standard deviation of concentration data from POCIS exposed in triplicates varied between approx. 5 %-50 %. Utilizing exploratory data analysis approach and tools enabled us to obtain a relatively complex picture of the situation and progress regarding pesticide pollution of groundwater in the monitored areas.

Opportunistic consumption of marine pelagic, terrestrial, and chemosynthetic organic matter by macrofauna on the Arctic shelf: a stable isotope approach.

Macrofauna can contribute substantially to the organic matter cycling on the seafloor, yet the role of terrestrial and chemosynthetic organic matter in the diets of microphagous (deposit and suspension) feeders is poorly understood. In the present study, we used stable isotopes of carbon and nitrogen to test the hypothesis that the terrestrial organic matter supplied with river runoff and local chemosynthetic production at methane seeps might be important organic matter sources for macrofaunal consumers on the Laptev Sea shelf. We sampled locations from three habitats with the presumed differences in organic matter supply: "Delta" with terrestrial inputs from the Lena River, "Background" on the northern part of the shelf with pelagic production as the main organic matter source, and "Seep" in the areas with detected methane seepage, where chemosynthetic production might be available. Macrobenthic communities inhabiting each of the habitats were characterized by a distinct isotopic niche, mostly in terms of δ13C values, directly reflecting differences in the origin of organic matter supply, while δ15N values mostly reflected the feeding group (surface deposit/suspension feeders, subsurface deposit feeders, and carnivores). We conclude that both terrestrial and chemosynthetic organic matter sources might be substitutes for pelagic primary production in the benthic food webs on the largely oligotrophic Laptev Sea shelf. Furthermore, species-specific differences in the isotopic niches of species belonging to the same feeding group are discussed, as well as the isotopic niches of the symbiotrophic tubeworm Oligobrachia sp. and the rissoid gastropod Frigidoalvania sp., which are exclusively associated with methane seeps.

Production of Fluorescent Dissolved Organic Matter by Microalgae Strains from the Ob and Yenisei Gulfs (Siberia).

Dissolved organic matter (DOM) is an important component of aquatic environments; it plays a key role in the biogeochemical cycles of many chemical elements. Using excitation-emission matrix fluorescence spectroscopy, we examined the fluorescent fraction of DOM (FDOM) produced at the stationary phase of growth of five strains of microalgae sampled and isolated from the Ob and Yenisei gulfs. Based on the morphological and molecular descriptions, the strains were identified as diatoms (Asterionella formosa, Fragilaria cf. crotonensis, and Stephanodiscus hantzschii), green microalgae (Desmodesmus armatus), and yellow-green microalgae (Tribonema cf. minus). Three fluorescent components were validated in parallel factor analysis (PARAFAC): one of them was characterized by protein-like fluorescence (similar to peak T), two others, by humic-like fluorescence (peaks A and C). The portion of fluorescence intensity of humic compounds (peak A) to the total fluorescence intensity was the lowest (27 ± 5%) and showed little variation between species. Protein-like fluorescence was most intense (45 ± 16%), but along with humic-like fluorescence with emission maximum at 470 nm (28 ± 14%), varied considerably for different algae strains. The direct optical investigation of FDOM produced during the cultivation of the studied algae strains confirms the possibility of autochthonous production of humic-like FDOM in the Arctic shelf regions.

Brønsted acidic ionic liquid-catalyzed tandem reaction: an efficient and sustainable approach towards the regioselective synthesis and molecular docking studies of 4-hydroxycoumarin-substituted indoles bearing lower E-factors.

1-Butane sulfonic acid-3-methylimidazolium tosylate, [BSMIM]OTs, is a remarkable catalyst for the cascade synthesis of coumarin-functionalized indole derivatives via a tandem cyclization reaction of aniline and phenylglyoxal monohydrate. This reaction possibly proceeds through imine formation/nucleophilic addition/cyclization. In addition, this method shows lower E-factors. A clean reaction, easily accessible reactants, metal-free and environmentally friendly reaction conditions, and reusability of the catalyst are the notable advantages of this procedure. In addition, molecular docking studies show the theoretical possibility of binding these types of synthesized compounds to key proteins in tumorigenesis.

Towards novel tacrine analogues: Pd(dppf)Cl2·CH2Cl2 catalyzed improved synthesis, in silico docking and hepatotoxicity studies.

A plethora of 6-(hetero)aryl C-C and C-N bonded tacrine analogues has been made accessible by employing palladium mediated (Suzuki-Miyaura, Heck, Sonogashira, Stille and Buchwald) cross-coupling reactions, starting from either halogenated or borylated residues. The successful use of Pd(dppf)Cl2·CH2Cl2 as a common catalytic system in realizing all these otherwise challenging transformations is the highlight of our optimized protocols. The analogues thus synthesized allow the available chemical space around the C-6 of this biologically relevant tacrine core to be explored. The in silico docking studies of the synthesized compounds were carried out against the acetylcholinesterase (AChE) enzyme. The hepatotoxicity studies of these compounds were done against complexes of CYP1A2 and CYP3A4 proteins with known inhibitors like 7,8-benzoflavone and ketoconazole, respectively.

Repolarizing potassium currents in working myocardium of Japanese quail: a novel translational model for cardiac electrophysiology.

Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (IKr, IKs, IKur and Ito). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents IKr, IKs and Ito - the latter was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 µmol l-1), HMR 1556 (30 µmol l-1) and 4-aminopyridine (3 mmol l-1), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates.

Two known and one new species of Draconematidae and Epsilonematida (Nematoda, Desmodorida) from the White Sea, North Russia.

Morphological descriptions of three "walking nematode" species found for the first time in the White Sea are presented. Draconema ophicephalum (Claparède, 1863) (Draconematidae) and Epsilonema steineri Chitwood, 1935 (Epsilonematidae), both known from insufficient material and females only, are re-described and problems of their taxonomic identification as well as species compositions of respective genera are discussed. The new species Prochaetosoma marisalbi sp. n. (Draconematidae) differs from other Prochaetosoma species except P. longicapitatum (Allgén, 1935) in that the pharyngeal bulb lumen is not cuticularised, from P. longicapitatum by shape of body and rostrum, greater number of cephalic adhesive tubes, and from P. maertensi Decraemer, 1989 by having a relatively longer tail, fewer anterior adhesive tubes and longer spicules, besides lacking cuticular thickening in the pharyngeal bulb. Draconema hoonsooi, D. youngeouni, P.rochaetosoma beomseomense, P. brevicaudatum, P. byungilli, P. cracense, P. saheungi, P. sujungi, P. supseomense erected by Rho & Min (2011) are considered as invalid species while Prochaetosoma arcticum, P. lugubre and Epsilonema cygnoides are assumed as species inquirenda. From a phylogenetic tree based on the 18S rRNA gene, all three White Sea species adjoin to unidentified species of their respective genera.

Genomic study of the Ket: a Paleo-Eskimo-related ethnic group with significant ancient North Eurasian ancestry.

The Kets, an ethnic group in the Yenisei River basin, Russia, are considered the last nomadic hunter-gatherers of Siberia, and Ket language has no transparent affiliation with any language family. We investigated connections between the Kets and Siberian and North American populations, with emphasis on the Mal'ta and Paleo-Eskimo ancient genomes, using original data from 46 unrelated samples of Kets and 42 samples of their neighboring ethnic groups (Uralic-speaking Nganasans, Enets, and Selkups). We genotyped over 130,000 autosomal SNPs, identified mitochondrial and Y-chromosomal haplogroups, and performed high-coverage genome sequencing of two Ket individuals. We established that Nganasans, Kets, Selkups, and Yukaghirs form a cluster of populations most closely related to Paleo-Eskimos in Siberia (not considering indigenous populations of Chukotka and Kamchatka). Kets are closely related to modern Selkups and to some Bronze and Iron Age populations of the Altai region, with all these groups sharing a high degree of Mal'ta ancestry. Implications of these findings for the linguistic hypothesis uniting Ket and Na-Dene languages into a language macrofamily are discussed.

Mitochondrial genome of two marine fungal species.

Here is a first record for a mitochondrial genome of marine fungus Acremonium fuci and closely related species Emericellopsis sp. One strain for each species, differentiated by morphological features was studied. Complete mitochondrial sequences are 24 565 and 24 951 bp in length. The circular molecule encodes all genes typical for fungal mitochondrial genomes: 15 protein-coding genes, 28 tRNAs, and large and small subunits of RNA. All structural genes are located on one strand and transcribed in one direction. Mitogenomes of species have 99% identity in protein-coding genes, but differ in some structural features: one of them has additional ORF with a similarity to cox1 gene, and another one has an intron in nad5 gene.

Carbon monoxide modulates electrical activity of murine myocardium via cGMP-dependent mechanisms.

Carbon monoxide (CO) is critical in cell signaling, and inhalation of gaseous CO can impact cardiovascular physiology. We have investigated electrophysiological effects of CO and their potential cGMP-dependent mechanism in isolated preparations of murine myocardium. The standard microelectrode technique was used to record myocardial action potentials (APs). Exogenous CO (0.96 × 10(-4)-4.8 × 10(-4) M) decreased AP duration in atrial and ventricular tissue and accelerated pacemaking activity in sinoatrial node. Inhibitors of heme oxygenases (zinc and tin protoporphyrin IX), which are responsible for endogenous CO production, induced the opposite effects. Inhibitor of soluble guanylate cyclase (sGC), ODQ (10(-5) M) halved CO-induced AP shortening, while sGC activator azosidnone (10(-5) M-3 × 10(-4) M) and cGMP analog BrcGMP (3 × 10(-4) M) induced the same effects as CO. To see if CO effects are attributed to differential regulation of phosphodiesterase 2 (PDE2) and 3 (PDE3), we used inhibitors of these enzymes. Milrinone (2 × 10(-6) M), selective inhibitor of cGMP-downregulated PDE3, blocked CO-induced rhythm acceleration. EHNA(2 × 10(-6) M), which inhibits cGMP-upregulated PDE2, attenuated CO-induced AP shortening, but failed to induce any positive chronotropic effect. Our findings indicate that PDE2 activity prevails in working myocardium, while PDE3 is more active in sinoatrial node. The results suggest that cardiac effects of CO are at least partly attributed to activation of sGC and subsequent elevation of cGMP intracellular content. In sinoatrial node, this leads to PDE3 inhibition, increased cAMP content, and positive chronotropy, while it also causes PDE2 stimulation in working myocardium, thereby enhancing cAMP degradation and producing AP shortening. Thus, CO induces significant alterations of cardiac electrical activity via cGMP-dependent mechanism and should be considered as a novel regulator of cardiac electrophysiology.

Thin-layer chromatographic plates with monolithic layer of silica: production, physical-chemical characteristics, separation capabilities.

The technique for production of thin-layer chromatographic plates with fixed monolithic layer of sorbent was developed on the basis of investigation of factors affecting sorption capacity, sorption kinetics and mechanical stability of monoliths. The optimal reaction mixture for sol-gel synthesis of monoliths consisted of tetraethoxysilane, buffer solution with pH 7.4, N,N-dimethylformamide, ethanol, polyethyleneglycol with molecular weight 1000 and cetylpyridinium chloride in molar ratio 1.0:4.6:1.4:7.6:0.26:8×10(-3). On the basis of analysis of sorption kinetics of malachite green on the monoliths it was concluded that mechanism of sorption includes chemisorption. The optimized conditions for fixing the monolithic layer on the carrier and its drying allow obtaining undisturbed monolithic layer, which was used for test mixtures separation. The increase of monolithic layer thickness in comparison with ultrathin-layer chromatographic plates allows detecting visually at reasonable concentrations and loaded sample volumes the spots of food and synthetic dyes.

Novel highly charged silica-coated Tb(III) nanoparticles with fluorescent properties sensitive to ion exchange and energy transfer processes in aqueous dispersions.

Novel silica-coated Tb(III) nanoparticles with high luminecsence were synthesized using the reverse microemulsion procedure. The quenching of luminescent properties of these nanoparticles can be achieved by ion exchange and energy transfer mechanisms. The quenching through the ion exchange of Tb(III) by H+ or La(III) is time dependent, indicating that the ion exchange is probably diffusion controlled. The quenching by Co(III) complex cations is achieved by the energy transfer mechanism and thus is not time dependent. The analysis of quenching data in Stern-Volmer cooordinates reveal the negative charge of the silica-coated Tb(III)-TCAS nanoparticles and several types of luminophoric species, located within the core and close to the surface of silica nanoparticles.

Quartz crystal microbalance immunosensor for the detection of antibodies to double-stranded DNA.

We report the development of a novel quartz crystal microbalance immunosensor with the simultaneous measurement of resonance frequency and motional resistance for the detection of antibodies to double-stranded DNA (dsDNA). The immobilization of poly(L-lysine) and subsequent complexation with DNA resulted in formation of a sensitive dsDNA-containing nanofilm on the surface of a gold electrode. Atomic force microscopy has been applied for the characterization of a poly(L-lysine)-DNA film. After the blocking with bovine serum albumin, the immunosensor in flow-injection mode was used to detect the antibodies to dsDNA in purified protein solutions of antibodies to dsDNA and to single-stranded DNA, monoclonal human immunoglobulin G, DNase I and in blood serum of patients with bronchial asthma and systemic lupus erythematosus. Experimental results indicate high sensitivity and selectivity of the immunosensor.