Atomic force microscopy of spherical intermediates on the pathway to fibril formation of influenza A virus nuclear export protein.

The nuclear export protein of the influenza A virus (NEP) is involved in many important processes of the virus life cycle. This makes it an attractive target for the treatment of a disease caused by a virus. Previously it has been shown, that recombinant variants of NEP are highly prone to aggregation in solution under various conditions with the formation of amyloid-like aggregates. In the present work, the amyloid nature of NEP aggregates was evidenced by Congo red binding assays. Atomic force microscopy has shown that NEP can form two types of spherical nanoparticles, which provide an alternative pathway for the formation of amyloid-like fibrils. Type I of these "fibrillogenic" spheres, formed under physiological conditions, represents the micelle-like particles with height 10-60 nm, which can generate worm-like flexible fibrils with the diameter 2.5-4.0 nm, length 20-500 nm and the Young's modulus ~73 MPa. Type II spherical aggregates with size of about 400-1000 nm, formed at elevated temperatures, includes fractions of drop-like and vesicle-like particles, generating more rigid amyloid-like fibrils with height of ~8 nm, and length of up to 2 µm. The hypothetical mechanism of fibril formation via nanospherical structures was suggested. RESEARCH HIGHLIGHTS: AFM has revealed two types of the influenza A virus nuclear export protein spherical aggregates. They provide an alternative pathway for the formation of amyloid-like fibrils. The mechanism of fibril formation via spherical structures is suggested.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated "symplastic" phloem loaders, as opposed to "apoplastic" loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

GTR-Mediated Radial Import Directs Accumulation of Defensive Glucosinolates to Sulfur-Rich Cells in the Phloem Cap of Arabidopsis Inflorescence Stem.

In the phloem cap region of Arabidopsis plants, sulfur-rich cells (S-cells) accumulate >100 mM glucosinolates (GLS), but are biosynthetically inactive. The source and route of S-cell-bound GLS remain elusive. In this study, using single-cell sampling and scanning electron microscopy with energy-dispersive X-ray analysis we show that two GLS importers, NPF2.10/GTR1 and NPF2.11/GTR2, are critical for GLS accumulation in S-cells, although they are not localized in the S-cells. Comparison of GLS levels in S-cells in multiple combinations of homo- and heterografts of gtr1 gtr2, biosynthetic null mutant and wild-type plants indicate that S-cells accumulate GLS via symplasmic connections either directly from neighboring biosynthetic cells or indirectly to non-neighboring cells expressing GTR1/2. Distinct sources and transport routes exist for different types of GLS, and vary depending on the position of S-cells in the inflorescence stem. Based on these findings, we propose a model illustrating the GLS transport routes either directly from biosynthetic cells or via GTR-mediated import from apoplastic space radially into a symplasmic domain, wherein the S-cells are the ultimate sink. Similarly, we observed accumulation of the cyanogenic glucoside defensive compounds in high-turgor cells in the phloem cap of Lotus japonicus, suggesting that storage of defensive compounds in high-turgor cells may be a general mechanism for chemical protection of the phloem cap.

Vibrational spectroscopy and density of K2O-B2O3-GeO2 glasses with variable B/Ge ratio.

Glasses of the K2O-B2O3-GeO2 system were studied by means of Raman and IR spectroscopy. The density of the samples was measured and the dependence of the molar volume and atomic density on composition was calculated. Curve-fitting of Raman spectra was applied to obtain a definition of the main structural units formed in the system. The conditions for highly-coordinated boron and germanium atoms were obtained. It was shown that potassium cations remain connected to germanate structural units at a B/Ge ratio of up to 1, whereas the explicit redistribution of borate and germanate structural groupings becomes most noticeable only at a B/Ge ratio > 2.

Hypervelocity collision and water-rock interaction in space preserved in the Chelyabinsk ordinary chondrite.

A comprehensive geochemical study of the Chelyabinsk meteorite reveals further details regarding its history of impact-related fragmentation and melting, and later aqueous alteration, during its transit toward Earth. We support an ∼30 Ma age obtained by Ar-Ar method (Beard et al., 2014) for the impact-related melting, based on Rb-Sr isotope analyses of a melt domain. An irregularly shaped olivine with a distinct O isotope composition in a melt domain appears to be a fragment of a silicate-rich impactor. Hydrogen and Li concentrations and isotopic compositions, textures of Fe oxyhydroxides, and the presence of organic materials located in fractures, are together consistent with aqueous alteration, and this alteration could have pre-dated interaction with the Earth's atmosphere. As one model, we suggest that hypervelocity capture of the impact-related debris by a comet nucleus could have led to shock-wave-induced supercritical aqueous fluids dissolving the silicate, metallic, and organic matter, with later ice sublimation yielding a rocky rubble pile sampled by the meteorite.

An in vitro and in silico study on the antioxidant and cell culture-based study on the chemoprotective activities of fish muscle protein hydrolysates obtained from European seabass and gilthead seabream.

European seabass (Dicentrarchus labrax, Linnaeus, 1758) (L) and gilthead seabream (Sparus aurata, Linnaeus, 1758) (C) muscles were hydrolysated by Alcalase (Lalc, Calc) and Chymotrypsin (Lch, Cch) then hydrolysates were examined and their peptide profiles obtained. A total of 765, 794, 132 and 232 peptides were identified in Calc, Lalc, Cch and Lch, respectively. Although, Lch and Cch were expected to have more antioxidant capacity because of their peptide profiles, Alcalase hydrolysates observed in vitro, were slightly higher (TEAC assay for Calc: 848.11 ±â€¯60.78 µmol TE/g protein). Maximum inhibition of oxidative stress was determined for Lalc (12.8% ±â€¯4.5%) in MDCK1 cell lines. Highest proliferative capacity observed for Calc (147.0% ±â€¯3.1%) at MTT assay in MDCK1 cell culture. Lch showed the highest chemopreventive effect with a 40-60% decrease for human colon adenocarcinoma cell line HT-29. This research points out the importance of aquatic sources as raw materials for peptide researches.

The structure of iPLA2ß reveals dimeric active sites and suggests mechanisms of regulation and localization.

Calcium-independent phospholipase A2ß (iPLA2ß) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson's disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2ß-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2ß that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA2ß cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA2ß in the brain.

The exon junction complex senses energetic stress and regulates contractility and cell architecture in cardiac myocytes.

The exon junction complex (EJC) is the main mechanism by which cells select specific mRNAs for translation into protein. We hypothesized that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes, with implications for the failing heart. In cultured rat neonatal myocytes, we examined the cellular distribution of two EJC components eukaryotic translation initiation factor 4A isoform 3 (eIF4A3) and mago nashi homologue (Mago) in response to metabolic stress. There was significant relocalization of eIF4A3 and Mago from the nucleus to cytoplasm following 18 h of hypoxia. Treating myocytes with 50 mM NaN3 for 4 h to mimic the metabolic stress induced by hypoxia also resulted in significant relocalization of eIF4A3 and Mago to the cytoplasm. To examine whether the effects of metabolic stress on the EJC proteins were dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1 µM dorsomorphin (DM) in combination with NaN3 DM augmented the translocation of Mago and eIF4A3 from the nucleus to the cytoplasm. Knockdown of eIF4A3 resulted in cessation of cell contractility 96 h post-treatment and a significant reduction in the number of intact sarcomeres. Cell area was significantly reduced by both hypoxia and eIF4A3 knockdown, whilst eIF4A3 knockdown also significantly reduced nuclear size. The reduction in nuclear size is unlikely to be related to apoptosis as it was reversed in combination with hypoxia. These data suggest for the first time that eIF4A3 and potentially other EJC members play an important role in the myocyte stress response, cell contractility and morphology.

Level of Granzyme B-positive T-regulatory cells is a strong predictor biomarker of acute Graft-versus-host disease after day +30 after allo-HSCT.

Acute Graft-versus-host-disease (aGVHD), the major complication and one of the main causes of poor outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays there are no widely accepted cell, plasma or another biomarker that can be used for aGVHD prediction. We hypothesized that a level of Granzyme B-positive T regulatory (GZMB-positive Treg) cells on day+30 after allo-HSCT could be the measure of immune response suppression and could predict aGVHD development after day +30. We applied a widespread and easy-to-perform method of multicolor flow cytometry to measure level of GZMB-positive Treg cells. Levels of GZMB-positive Tregs on day +30 after allo-HSCT were significantly higher in those patients who never developed aGVHD in comparison with the other group of patient with aGVHD after day +30 (p=0.0229). We conclude that the level of GZMB-positive Treg cells is a strong predictor of acute Graft-versus-host disease after day +30 after allo-HSCT.

A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes.

Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2∼Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s.

Laccase multigene families in Agaricomycetes.

Here we present the results of the exploration of laccase multigene families (MGFs) in basidiomycetous fungi from different taxonomic groups using a next generation sequencing (NGS) technology. In our study, multiple laccase genes were identified in all of the investigated fungi (13 species) from Polyporaceae, Phanerochaetaceae, Meruliaceae, Pleurotaceae, Physalacriaceae, and Peniophoraceae families. It was shown that phylogenetic positioning of the newly identified sequences exhibit patterns of clusterization with respect to enzyme properties. This can be a potentially useful tool for selecting naturally existing laccases with different physicochemical characteristics relevant to different biotechnological applications. Moreover, the method developed in this study can be used in the screening of environmental samples and fast characterization of laccase MGFs in newly identified fungal species.

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters.

BACKGROUND: Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood. METHODS: The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM). RESULTS: In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance. CONCLUSIONS: The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one. GENERAL SIGNIFICANCE: The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.

Extracellular proteins of Trametes hirsuta st. 072 induced by copper ions and a lignocellulose substrate.

BACKGROUND: Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment. RESULTS: This study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attributed the observed extracellular LiP - like activity to the expressed versatile peroxidase (VP). An accessory enzyme, glyoxal oxidase, was found among the proteins secreted in the media during submerged cultivation of T. hirsuta both in the presence and in the absence of copper. However, aryl-alcohol oxidase (AAO) was not identified, despite the presence of AAO enzymatic activity secreted by the fungus. The spectra of the expressed enzymes dramatically changed depending on the growth conditions. Transfer from submerged cultivation to surface cultivation with the lignocellulose substrate switched off expression of exo-ß-1,3-glucanase and α-amylase and turned on secretion of endo-ß-1,3-glucanase and a range of glycosidases. In addition, an aspartic peptidase started being expressed instead of family S53 protease. For the first time, we report production of cerato-platanin proteins by Trametes species. The secretion of cerato-platanins was observed only in response to contact with lignocellulose, thus indicating a specific role of these proteins in degradation of the lignocellulose substrates. CONCLUSIONS: Our results suggest a sequential mechanism of natural substrate degradation by T. hirsuta, in which the fungus produces different sets of enzymes to digest all main components of the substrate during cultivation.

Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 µM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-µM and 10-µM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

Draft Genome Sequence of the Fungus Trametes hirsuta 072.

A standard draft genome sequence of the white rot saprotrophic fungus Trametes hirsuta 072 (Basidiomycota, Polyporales) is presented. The genome sequence contains about 33.6 Mb assembled in 141 scaffolds with a G+C content of ~57.6%. The draft genome annotation predicts 14,598 putative protein-coding open reading frames (ORFs).

Structure-Functional Study of Tyrosine and Methionine Dipeptides: An Approach to Antioxidant Activity Prediction.

Quantum chemical methods allow screening and prediction of peptide antioxidant activity on the basis of known experimental data. It can be used to design the selective proteolysis of protein sources in order to obtain products with antioxidant activity. Molecular geometry and electronic descriptors of redox-active amino acids, as well as tyrosine and methionine-containing dipeptides, were studied by Density Functional Theory method. The calculated data was used to reveal several descriptors responsible for the antioxidant capacities of the model compounds based on their experimentally obtained antioxidant capacities against ABTS (2,2'-Azino-bis-(3-ethyl-benzothiazoline-6-sulfonate)) and peroxyl radical. A formula to predict antioxidant activity of peptides was proposed.

The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction.

Laccases, blue copper-containing oxidases, ≿ an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072, an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed.

Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes.

Diacylglyceryltrimethylhomoserines (DGTS) are betaine-type lipids that are phosphate-free analogs of phosphatidylcholines (PC). DGTS are abundant in some bacteria, algae, primitive vascular plants and fungi. In this study, we report inorganic phosphate (Pi) deficiency-induced DGTS synthesis in the basidial fungus Flammulina velutipes (Curt.: Fr.) Sing. We present results of an expression analysis of the BTA1 gene that codes for betaine lipid synthase and two genes of PC biosynthesis (CHO2 and CPT1) during phosphate starvation of F. velutipes culture. We demonstrate that FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite depletion in PC, both CHO2 and CPT1 were determined to have increased expression. We also describe the deduced amino acid sequence and genomic structure of the BTA1 gene in F. velutipes. Phylogenetic relationships between putative orthologs of BTA1 proteins of basidiomycete fungi are discussed.