Titanium dioxide nanoparticles temporarily influence the sea urchin immunological state suppressing inflammatory-relate gene transcription and boosting antioxidant metabolic activity.

Abstract Titanium dioxide nanoparticles (TiO2NPs) are revolutionizing biomedicine due to their potential application as diagnostic and therapeutic agents. However, the TiO2NP immune-compatibility remains an open issue, even for ethical reasons. In this work, we investigated the immunomodulatory effects of TiO2NPs in an emergent proxy to human non-mammalian model for in vitro basic and translational immunology: the sea urchin Paracentrotus lividus. To highlight on the new insights into the evolutionarily conserved intracellular signaling and metabolism pathways involved in immune-TiO2NP recognition/interaction we applied a wide-ranging approach, including electron microscopy, biochemistry, transcriptomics and metabolomics. Findings highlight that TiO2NPs interact with immune cells suppressing the expression of genes encoding for proteins involved in immune response and apoptosis (e.g. NF-κB, FGFR2, JUN, MAPK14, FAS, VEGFR, Casp8), and boosting the immune cell antioxidant metabolic activity (e.g. pentose phosphate, cysteine-methionine, glycine-serine metabolism pathways). TiO2NP uptake was circumscribed to phagosomes/phagolysosomes, depicting harmless vesicular internalization. Our findings underlined that under TiO2NP-exposure sea urchin innate immune system is able to control inflammatory signaling, excite antioxidant metabolic activity and acquire immunological tolerance, providing a new level of understanding of the TiO2NP immune-compatibility that could be useful for the development in Nano medicines

Permeable membranes PUR/TETA and PUR/TEPA for CO2 capture prepared with one-step electrospinning technology

A simple one-step technology of wire electrospinning is presented for the manufacturing of air-permeable CO2-capturing membranes, easily transferable to industrial production lines. The design of the chemically-modified polyurethane nanofiber membranes for CO2 capture was based on a combination of molecular modeling and technological experiments using one-step electrospinning (i.e., a modifying agent dissolved directly in a spinning solution). Polyurethane (PUR Larithane), chemically modified by TETA/TEPA amines, was used in the present study for the membrane design. Special attention was paid to two key parameters significant for the design of the functional unit, i.e., the CO2 sorption capacity and air permeability which depended on the amine concentration. The optimal combination of these parameters was found for the PUR/TEPA membrane (5 wt.% of TEPA in spinning solution): the sorption capacity was 13.97 cm(3)/g with an air permeability of 0.020 m/s. Molecular modeling proved to be a valuable tool that helped to clarify, at the molecular level, the structure of chemically-modified nanofibrous membranes.Web of Science1011art. no. 10

Soil Matrix Determines the Outcome of Interaction Between Mycorrhizal Symbiosis and Biochar for Andropogon gerardii Growth and Nutrition

Biochar has been heralded as a multipurpose soil amendment to sustainably increase soil fertility and crop yields, affect soil hydraulic properties, reduce nutrient losses, and sequester carbon. Some of the most spectacular results of biochar (and organic nutrient) inputs are the terra preta soils in the Amazon, dark anthropogenic soils with extremely high fertility sustained over centuries. Such soil improvements have been particularly difficult to achieve on a short run, leading to speculations that biochar may need to age (weather) in soil to show its best. Further, interaction of biochar with arbuscular mycorrhizal fungi (AMF), important root symbionts of a great majority of terrestrial plants including most agricultural crops, remains little explored. To study the effect of aged biochar on highly mycotrophic Andropogon gerardii plants and their associated AMF, we made use of softwood biochar, collected from a historic charcoal burning site. This biochar (either untreated or chemically activated, the latter serving as a proxy for freshly prepared biochar) was added into two agricultural soils (acid or alkaline), and compared to soils without biochar. These treatments were further crossed with inoculation with a synthetic AMF community to address possible interactions between biochar and the AMF. Biochar application was generally detrimental for growth and mineral nutrition of our experimental plants, but had no effect on the extent of their root colonized by the AMF, nor did it affect composition of their root-borne AMF communities. In contrast, biochar affected development of two out of five AMF (Claroideoglomus and Funneliformis) in the soil. Establishment of symbiosis with AMF largely mitigated biochar-induced suppression of plant growth and mineral nutrition, mainly by improving plant acquisition of phosphorus. Both mycorrhizal and non-mycorrhizal plants grew well in the acid soil without biochar application, whereas non-mycorrhizal plants remained stunted in the alkaline soils under all situations (with or without biochar). These different and strong effects indicate that response of plants to biochar application are largely dependent on soil matrix and also on microbes such as AMF, and call for further research to enable qualified predictions of the effects of different biochar applications on field-grown crops and soil processes

Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

<p>Abstract</p> <p>Background</p> <p>Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.</p> <p>Results</p> <p>A nitrilase gene was amplified by PCR from the cDNA library of <it>Aspergillus niger </it>K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in <it>Escherichia coli </it>BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val₃₂₇- Asn₃₅₆) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp₂₉₈-Val₃₁₃peptide was shortened to Asp₂₉₈-Arg₃₁₀in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.</p> <p>Conclusions</p> <p>The nitrilase from <it>Aspergillus niger </it>K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in <it>Aspergillus </it>and <it>Penicillium </it>genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by <it>in vitro </it>protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.</p

Životní cyklus Streptomycet rostoucích na balotine sledovaný pomocí řádkovací elektronové mikroskopie

Here we present an easy methodological approach for studying the complex life cycle of Streptomycetes grown on glass beads (ballotina) by classical SEM. It is based on cultivation of Streptomycetes in mini Petri dishes made from glass scintillation vial

Phage Tail-Like (High-Molecular-Weight) Bacteriocins of Budvicia aquatica and Pragia fontium (Enterobacteriaceae)

Electron microscopic analysis of contractile phage tail-like bacteriocins of three Pragia fontium strains and one Budvicia aquatica strain was performed. Fonticin and aquaticin are remarkably heat sensitive but trypsin resistant. Simultaneous production of contractile and flexible phage tail-like bacteriocins in the P. fontium 64613 strain is shown for the first time

Secondary Metabolites Produced during the Germination of Streptomyces coelicolor

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments

Continuous Diastereomeric Kinetic Resolution—Silybins A and B

The natural diastereomeric mixture of silybins A and B is often used (and considered) as a single flavonolignan isolated from the fruit extract of milk thistle (Silybum marianum), silymarin. However, optically pure silybin diastereomers are required for the evaluation of their biological activity. The separation of silybin diastereomers by standard chromatographic methods is not trivial. Preparative chemoenzymatic resolution of silybin diastereomers has been published, but its optimization and scale-up are needed. Here we present a continuous flow reactor for the chemoenzymatic kinetic resolution of silybin diastereomers catalyzed by Candida antarctica lipase B (CALB) immobilized on acrylic resin beads (Novozym® 435). Temperature, flow rate, and starting material concentration were varied to determine optimal reaction conditions. The variables observed were conversion and diastereomeric ratio. Optimal conditions were chosen to allow kilogram-scale reactions and were determined to be −5 °C, 8 g/L silybin, and a flow rate of 16 mL/min. No significant carrier degradation was observed after approximately 30 cycles (30 days). Under optimal conditions and using a 1000 × 15 mm column, 20 g of silybin per day can be easily processed, yielding 6.7 and 5.6 g of silybin A and silybin B, respectively. Further scale-up depends only on the size of the reactor

Differential Effects of the Flavonolignans Silybin, Silychristin and 2,3-Dehydrosilybin on <i>Mesocestoides vogae</i> Larvae (Cestoda) under Hypoxic and Aerobic In Vitro Conditions

Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans&#8212;silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)&#8212;on M. vogae larvae at concentrations of 5 and 50 &#956;M under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 &#956;M, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 &#956;M concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism