Impact of Biogenic and Chemogenic Selenium Nanoparticles on Model Eukaryotic Lipid Membranes.

Microbial nanotechnology is an expanding research area devoted to producing biogenic metal and metalloid nanomaterials (NMs) using microorganisms. Often, biogenic NMs are explored as antimicrobial, anticancer, or antioxidant agents. Yet, most studies focus on their applications rather than the underlying mechanism of action or toxicity. Here, we evaluate the toxicity of our well-characterized biogenic selenium nanoparticles (bSeNPs) produced by the Stenotrophomonas maltophilia strain SeITE02 against the model yeast Saccharomyces cerevisiae comparing it with chemogenic SeNPs (cSeNPs). Knowing from previous studies that the biogenic extract contained bSeNPs in an organic material (OM) and supported here by Fourier transform infrared spectroscopy, we removed and incubated it with cSeNPs (cSeNPs_OM) to assess its influence on the toxicity of these formulations. Specifically, we focused on the first stages of the eukaryotic cell exposure to these samples─i.e., their interaction with the cell lipid membrane, which was mimicked by preparing vesicles from yeast polar lipid extract or phosphatidylcholine lipids. Fluidity changes derived from biogenic and chemogenic samples revealed that the bSeNP extract mediated the overall rigidification of lipid vesicles, while cSeNPs showed negligible effects. The OM and cSeNPs_OM induced similar modifications to the bSeNP extract, reiterating the need to consider the OM influence on the physical-chemical and biological properties of bSeNP extracts.

Prokaryotic and eukaryotic traits support the biological role of the chloroplast outer envelope.

The plastid outer envelope (OE) is a mixture of components inherited from their prokaryotic ancestor like galactolipids, carotenoids and porin type ion channels supplemented with eukaryotic inventions to make the endosymbiotic process successful as well as to control plastid biogenesis and differentiation. In this review we wanted to highlight the importance of the OE proteins and its evolutionary origin. For a long time, the OE was thought to be a diffusion barrier only, but with the recent discoveries of all kinds of different proteins in the OE it has been shown that the OE can modulate various functions within the cell. The phenotypic changes show that channels like the outer envelope proteins OEP40, OEP16 or JASSY have a pronounced ion selectivity that cannot be replaced by other ion channels present in the OE. Eukaryotic additions, like the GTPase receptors Toc33 and Toc159 or the ubiquitin proteasome system for chloroplast protein quality control, round up the profile of the OE.

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches.

We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 µM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.

Bacterial, archaeal and micro-eukaryotic communities characterize a disease-suppressive or conducive soil and a cultivar resistant or susceptible to common scab.

Control of common scab disease can be reached by resistant cultivars or suppressive soils. Both mechanisms are likely to translate into particular potato microbiome profiles, but the relative importance of each is not known. Here, microbiomes of bulk and tuberosphere soil and of potato periderm were studied in one resistant and one susceptible cultivar grown in a conducive and a suppressive field. Disease severity was suppressed similarly by both means yet, the copy numbers of txtB gene (coding for a pathogenicity determinant) were similar in both soils but higher in periderms of the susceptible cultivar from conducive soil. Illumina sequencing of 16S rRNA genes for bacteria (completed by 16S rRNA microarray approach) and archaea, and of 18S rRNA genes for micro-eukarytes showed that in bacteria, the more important was the effect of cultivar and diversity decreased from resistant cultivar to bulk soil to susceptible cultivar. The major changes occurred in proportions of Actinobacteria, Chloroflexi, and Proteobacteria. In archaea and micro-eukaryotes, differences were primarily due to the suppressive and conducive soil. The effect of soil suppressiveness × cultivar resistance depended on the microbial community considered, but differed also with respect to soil and plant nutrient contents particularly in N, S and Fe.

A novel functional role of nickel in sperm motility and eukaryotic cell growth.

BACKGROUND: Metal ions are essential for numerous life processes. This study aims to investigate the relationship between seminal quality and ion levels in seminal plasma. BASIC PROCEDURES: A total of 205 semen samples were collected and seminal plasma ion levels were examined with inductively-coupled plasma-mass spectrometry. The nickel function was demonstrated by in vitro assay and cell growth. MAIN FINDINGS: The low sperm motility group showed distinctively reduced nickel concentration in seminal plasma compared with the normal sperm motility group. However, arsenic, sulfur, selenium, magnesium and zinc were negatively associated with sperm quality. No significant relationship between other examined cations and semen quality was observed. In vitro assay suggested low concentration of nickel significantly increased sperm total motility and progressive motility. Cell growth assay further confirmed nickel promoted eukaryotic yeast cell growth. Nickel level in seminal plasma may play important functions to determine sperm quality. PRINCIPAL CONCLUSIONS: Our study reveals a strong correlation between S, Mg, Se, Zn, As, Ni and seminal quality as well as discovers a novel functional role of nickel in sperm motility and eukaryotic cell growth. These findings may provide a potential avenue for assessment of sperm quality and treatment of reproduction disorders.

Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Graphene oxide significantly inhibits cell growth at sublethal concentrations by causing extracellular iron deficiency.

Graphene oxide (GO)-based materials are increasingly being used in medical materials and consumer products. However, their sublethal effects on biological systems are poorly understood. Here, we report that GO (at 10 to 160 mg/L) induced significant inhibitory effects on the growth of different unicellular organisms, including eukaryotes (i.e. Saccharomyces cerevisiae, Candida albicans, and Komagataella pastoris) and prokaryotes (Pseudomonas fluorescens). Growth inhibition could not be explained by commonly reported cytotoxicity mechanisms such as plasma membrane damage or oxidative stress. Based on transcriptomic analysis and measurement of extra- and intracellular iron concentrations, we show that the inhibitory effect of GO was mainly attributable to iron deficiency caused by binding to the O-functional groups of GO, which sequestered iron and disrupted iron-related physiological and metabolic processes. This inhibitory mechanism was corroborated with supplementary experiments, where adding bathophenanthroline disulfonate-an iron chelating agent-to the culture medium exerted similar inhibition, whereas removing surface O-functional groups of GO decreased iron sequestration and significantly alleviated the inhibitory effect. These findings highlight a potential indirect detrimental effect of nanomaterials (i.e. scavenging of critical nutrients), and encourage research on potential biomedical applications of GO-based materials to sequester iron and enhance treatment of iron-dependent diseases such as cancer and some pathogenic infections.

Ancient Systems of Sodium/Potassium Homeostasis as Predecessors of Membrane Bioenergetics.

Cell cytoplasm of archaea, bacteria, and eukaryotes contains substantially more potassium than sodium, and potassium cations are specifically required for many key cellular processes, including protein synthesis. This distinct ionic composition and requirements have been attributed to the emergence of the first cells in potassium-rich habitats. Different, albeit complementary, scenarios have been proposed for the primordial potassium-rich environments based on experimental data and theoretical considerations. Specifically, building on the observation that potassium prevails over sodium in the vapor of inland geothermal systems, we have argued that the first cells could emerge in the pools and puddles at the periphery of primordial anoxic geothermal fields, where the elementary composition of the condensed vapor would resemble the internal milieu of modern cells. Marine and freshwater environments generally contain more sodium than potassium. Therefore, to invade such environments, while maintaining excess of potassium over sodium in the cytoplasm, primordial cells needed means to extrude sodium ions. The foray into new, sodium-rich habitats was the likely driving force behind the evolution of diverse redox-, light-, chemically-, or osmotically-dependent sodium export pumps and the increase of membrane tightness. Here we present a scenario that details how the interplay between several, initially independent sodium pumps might have triggered the evolution of sodium-dependent membrane bioenergetics, followed by the separate emergence of the proton-dependent bioenergetics in archaea and bacteria. We also discuss the development of systems that utilize the sodium/potassium gradient across the cell membranes.

From zero to six double bonds: phospholipid unsaturation and organelle function.

Cellular phospholipids (PLs) differ by the nature of their polar heads as well as by the length and unsaturation level of their fatty acyl chains. We discuss how the ratio between saturated, monounsaturated, and polyunsaturated PLs impacts on the functions of such organelles as the endoplasmic reticulum, synaptic vesicles, and photoreceptor discs. Recent experiments and simulations suggest that polyunsaturated PLs respond differently to mechanical stress, including membrane bending, than monounsaturated PLs owing to their unique conformational plasticity. These findings suggest a rationale for PL acyl chain remodeling by acyltransferases and a molecular explanation for the importance of a balanced fatty acid diet.

Global transcriptome analysis of eukaryotic genes affected by gromwell extract.

BACKGROUND: Gromwell is known to have diverse pharmacological, cosmetic and nutritional benefits for humans. Nevertheless, the biological influence of gromwell extract (GE) on the general physiology of eukaryotic cells remains unknown. In this study a global transcriptome analysis was performed to identify genes affected by the addition of GE with Cryptococcus neoformans as the model system. RESULTS: In response to GE treatment, genes involved in signal transduction were immediately regulated, and the evolutionarily conserved sets of genes involved in the core cellular functions, including DNA replication, RNA transcription/processing and protein translation/processing, were generally up-regulated. In contrast, a number of genes involved in carbohydrate metabolism and transport, inorganic ion transport and metabolism, post-translational modification/protein turnover/chaperone functions and signal transduction were down-regulated. Among the GE-responsive genes that are also evolutionarily conserved in the human genome, the expression patterns of YSA1, TPO2, CFO1 and PZF1 were confirmed by northern blot analysis. Based on the functional characterization of some GE-responsive genes, it was found that GE treatment may promote cellular tolerance against a variety of environmental stresses in eukaryotes. CONCLUSIONS: GE treatment affects the expression levels of a significant portion of the Cryptococcus genome, implying that GE significantly affects the general physiology of eukaryotic cells.

The methionine sulfoxide reduction system: selenium utilization and methionine sulfoxide reductase enzymes and their functions.

SIGNIFICANCE: Selenium is utilized in the methionine sulfoxide reduction system that occurs in most organisms. Methionine sulfoxide reductases (Msrs), MsrA and MsrB, are the enzymes responsible for this system. Msrs repair oxidatively damaged proteins, protect against oxidative stress, and regulate protein function, and have also been implicated in the aging process. Selenoprotein forms of Msrs containing selenocysteine (Sec) at the catalytic site are found in bacteria, algae, and animals. RECENT ADVANCES: A selenoprotein MsrB1 knockout mouse has been developed. Significant progress in the biochemistry of Msrs has been made, which includes findings of a novel reducing system for Msrs and of an interesting reason for the use of Sec in the Msr system. The effects of mammalian MsrBs, including selenoprotein MsrB1 on fruit fly aging, have been investigated. Furthermore, it is evident that Msrs are involved in methionine metabolism and regulation of the trans-sulfuration pathway. CRITICAL ISSUES: This article presents recent progress in the Msr field while focusing on the physiological roles of mammalian Msrs, functions of selenoprotein forms of Msrs, and their biochemistry. FUTURE DIRECTIONS: A deeper understanding of the roles of Msrs in redox signaling, the aging process, and metabolism will be achieved. The identity of selenoproteome of Msrs will be sought along with characterization of the identified selenoprotein forms. Exploring new cellular targets and new functions of Msrs is also warranted.

Bactericidal activity against meticillin-resistant Staphylococcus aureus of a novel eukaryotic therapeutic recombinant antimicrobial peptide.

Antimicrobial peptides (AMPs) are one of several potential antibacterial agents in the current era of antibiotics facing severe challenges. In this study, the bactericidal activity and stability of two eukaryotic AMPs were determined. Both AMPs showed specific antibacterial activity in a HEK293T cell model infected with meticillin-resistant Staphylococcus aureus. The recombinant eukaryotic AMP pVAX1/hBD3-CBD showed better bactericidal activity and stability than the eukaryotic AMP pVAX1/hBD3. These results illustrate that this peptide, designed and used with eukaryotic expression and recombinant methods, should be studied and applied in further AMP research and trials.

Further analysis of Ames-negative rodent carcinogens that are only genotoxic in mammalian cells in vitro at concentrations exceeding 1 mM, including retesting of compounds of concern.

In the analysis by Parry et al. [Parry, J. M., Parry, E., Phrakonkham, P. and Corvi, R. (2010) Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing. Mutagenesis, 25, 531-538], 24 rodent carcinogens that were negative in the Ames test were identified that were only positive in mammalian cell tests at concentrations between 1 and 10 mM. These carcinogens can be subdivided into four groups as follows: (1) probable non-genotoxic (non-mutagenic) carcinogens, tumour promoters or negative for genotoxicity in vivo (n=10); (2) questionable carcinogens (n=4); (3) carcinogens with a probable genotoxic mode of action (n=5); (4) compounds where carcinogenicity or in vivo genotoxicity is unknown or unclear (n=5). It is not expected that in vitro mammalian cell tests should give positive results with Group 1 chemicals. Within Groups 2-4, five chemicals were considered a low priority because they could be detected using modified conditions because genotoxicity was associated with precipitate or pH shifts or because non-standard metabolism was required. The remaining nine chemicals were therefore considered most critical in terms of detection of genotoxic activity in mammalian cells. Daminozide was also included because it may have given positive responses between 1 and 10 mM. Many of the reported studies could have given positive results only at >1 mM because 'old' protocols were followed. These 10 chemicals have therefore been retested using modern protocols. Some were negative even up to 10 mM. Others were positive at concentrations <1 mM. Only methylolacrylamide was positive at a concentration >1 mM (2 mM = 202 µg/ml). Low-molecular weight substances may therefore require concentrations >1 mM, but further work is needed. Based on this analysis, it is concluded that the 10 mM upper limit in mammalian cell tests can be lowered without any loss of sensitivity in detecting genotoxic rodent carcinogens. A new limit of 1 mM or 500 µg/ml, whichever is the higher, is proposed.

Expression of acyl-lipid Delta12-desaturase gene in prokaryotic and eukaryotic cells and its effect on cold stress tolerance of potato.

We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

Anti-oxidative effects of Phellinus linteus and red ginseng extracts on oxidative stress-induced DNA damage.

Anti-oxidative effect of Phellinus linteus (P. linteus) and red ginseng extracts on DNA damage induced by reactive oxygen species (ROS) were investigated in this study. P. linteus (PLE) and red ginseng extracts (RGE) inhibited the breaking of E. coli ColE1 plasmid DNA strands as well as nuclear DNA of rat hepatocytes damaged by oxidative stress. In addition, a reaction mixture of PLE and RGE showed synergistic inhibitory effect against DNA damage. These results suggest that PLE and RGE have a cellular defensive effect against DNA damage induced by ROS.

Ursolic, oleanolic and betulinic acids: antibacterial spectra and selectivity indexes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA), three hydroxyl pentacyclic triterpenoic acids (HPTAs) naturally found in a large variety of vegetarian foods, medicinal herbs and plants have been investigated for antibacterial activity. AIM OF THE STUDY: To determine the antibacterial activity of UA, OA and BA, as well as the toxic impact on eukaryotic cells. MATERIALS AND METHODS: Minimum inhibitory concentrations were determined against five reference strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 & ATCC 29213, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853), as well as five antibiotic-resistant clinical isolates. Toxicity was evaluated against MRC-5 and HaCaT cell lines. RESULTS: No antibacterial activity was observed for BA; while OA and more particularly UA, did show a moderate to good antibacterial activity, but limited to Gram-positive bacteria. Nevertheless, OA and UA were devoid of antibacterial activities against clinical isolates. Moreover, viability and cytotoxic assays demonstrated that the three compounds induced a significant cytotoxicity. CONCLUSIONS: Despite of a relative similar chemical structure; UA, OA and BA harboured different antibacterial activities, with more significant ones for UA. However, considering both viability and toxicity values, these compounds seem to have a significant impact on eukaryotic cell viability.

Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation.

Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate- and glycerol-containing growth medium. Exponentially growing cells had a lipid content of 13% (w/w) of dry weight. A fattening of cells fed excess glycerol occurred in the post-exponential growth phase, after the medium was depleted of N or P. Lipid accumulation was also initiated by O2 limitation (below 1% of saturation). N starvation per se, or in combination with O2 limitation, gave the highest lipid content, i.e., 54% to 63% (w/w) of dry weight. The corresponding maximum culture density was 90 to 100 g/l dry biomass. The content of docosahexaenoic acid (22:6n-3) in N starved, well-oxygenated cells reached 29% (w/w) of total fatty acids but increased to 36% to 52% in O2-limited cells, depending on the time span of the limitation. O2-limited cells did not accumulate the monounsaturated fatty acids that were normally present. We inferred that the biological explanation is that O2 limitation hindered the O2-dependent desaturase(s) and favored the O2-independent polyunsaturated fatty acid synthase. The highest overall volumetric productivity of docosahexaenoic acid observed was 93 mg/l/h. Additionally, we present a protocol for quantitative lipid extraction, involving heat and protease treatment of freeze-dried thraustochytrids.

[Construction expression and biologic activity of recombinant human sCR1 eucaryotic cell].

AIM: To express human soluble complement receptor type 1(sCR1)protein using ferment cell secreting type carrier and study the extraorgan biologic activity of recombinant human sCR1 fusion protein. METHODS: Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained by RT-PCR and them, cloned into Pichia pastoris eukaryotic expression vector pPIC9k to construct the recombinant plasmid pPIC9k-sCR1 containing human sCR1.After identified by DNA sequencing, the recombinant plasmid pPIC9k-sCR1 was transformed into Pichia pastoris SMD1168. The ferment cell line of the recombinant sCR1 which was chosen by G418 resistance was identified by PCR, After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blot, purified by Ni(2+)-NTA agarose affinity chromatography, and its biologic activity was identified. RESULTS: The obtained Pichia pastoris secretion type yeast carrier pPIC9k-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant ferment cell line. The recombinant human sCR1 fusion protein was expressed by yeast cells containing pPIC9k-sCR1 induced by methanol. It was a protein band about M(r) 31 000 in gel, which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique. The highly purified sCR1 fusion protein and its biologic activity were detected obtained by Ni(2+)-NTA agarose affinity chromatography. CONCLUSION: The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastoris expression system, which resembles the human natural protein's antigenicity and biologic activity.

Eukaryotic selenoprotein synthesis: mechanistic insight incorporating new factors and new functions for old factors.

Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently.

Ediacaran oxidation and biotic evolution.

The link between the radiation of various lineages of eukaryotes in the latest Proterozoic and massive environmental changes--oxygenation, global ice ages and bolide impact--is the focus of much research interest. Fike et al. use carbon and sulphur isotope-chemostratigraphic data from Oman to propose three stages of oxidation in the Ediacaran oceans, and link the second and third stages to eukaryote diversification. The second stage, signalled by strongly 13C-depleted sedimentary carbonates (the 'Shuram excursion'), is believed to result from oxidation of a large, deep-ocean reservoir of organic carbon. Fike et al. use our data to assert that a correlative carbon isotope excursion in Australia coincided with the initial diversification of acanthomorphic acritarchs. Peak diversity is claimed to have coincided with subsequent deposition of 13C-enriched carbonate and the third oxidation stage. However, the authors seem to have misinterpreted our data, which instead indicate that diversification significantly preceded the Shuram excursion; this weakens their argument for a link between the inferred oxidation events and eukaryote evolution.