Comparison of sample preparation techniques for the physicochemical characterization of Orf virus particles.

The determination of the electrostatic charge of biological nanoparticles requires a purified, mono-disperse, and concentrated sample. Previous studies proofed an impact of the preparation protocol on the stability and electro-hydrodynamics of viruses, whereas commonly used methods are often complex and do not allow the required sample throughput. In the present study, the application of the (I) steric exclusion chromatography (SXC) for the Orf virus (ORFV) purification and subsequent physicochemical characterization was evaluated and compared to (II) SXC followed by centrifugal diafiltration and (III) sucrose cushion ultracentrifugation. The three methods were characterized in terms of protein removal, size distribution, infectious virus recovery, visual appearance, and electrophoretic mobility as a function of pH. All preparation techniques achieved a protein removal of more than 99 %, and (I) an infectious ORFV recovery of more than 85 %. Monodisperse samples were realized by (I) and (III). In summary, ORFV samples prepared by (I) and (III) displayed comparable quality. Additionally, (I) offered the shortest operation time and easy application. Based on the obtained data, the three procedures were ranked according to eight criteria of possible practical relevance, which delineate the potential of SXC as virus preparation method for physicochemical analysis.

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5'-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

A scalable downstream process for the purification of the cell culture-derived Orf virus for human or veterinary applications.

The large demand for safe and efficient viral vector-based vaccines and gene therapies against both inherited and acquired diseases accelerates the development of viral vectors. One outstanding example, the Orf virus, has a wide range of applications, a superior efficacy and an excellent safety profile combined with a reduced pathogenicity compared to other viral vectors. However, besides these favorable attributes, an efficient and scalable downstream process still needs to be developed. Recently, we screened potential chromatographic stationary phases for Orf virus purification. Based on these previous accomplishments, we developed a complete downstream process for the cell culture-derived Orf virus. The described process comprises a membrane-based clarification step, a nuclease treatment, steric exclusion chromatography, and a secondary chromatographic purification step using Capto® Core 700 resin. The applicability of this process to a variety of diverse Orf virus vectors was shown, testing two different genotypes. These studies render the possibility to apply the developed downstream scheme for both genotypes, and lead to overall virus yields of about 64 %, with step recoveries of >70 % for the clarification, and >90 % for the chromatography train. Protein concentrations of the final product are below the detection limits, and the final DNA concentration of about 1 ng per 1E + 06 infective virus units resembles a total DNA depletion of 96-98 %.

Global warming shifts the composition of the abundant bacterial phyllosphere microbiota as indicated by a cultivation-dependent and -independent study of the grassland phyllosphere of a long-term warming field experiment.

The leaf-colonizing bacterial microbiota was studied in a long-term warming experiment on a permanent grassland, which had been continuously exposed to increased surface temperature (+2°C) for more than six years. Two abundant plant species, Arrhenatherum elatius and Galium album, were studied. Surface warming reduced stomata opening and changed leaf metabolite profiles. Leaf surface colonization and the concentration of leaf-associated bacterial cells were not affected. However, bacterial 16S ribosomal RNA (rRNA) gene amplicon Illumina sequencing showed significant temperature effects on the plant species-specific phyllosphere microbiota. Warming partially affected the concentrations of cultured bacteria and had a significant effect on the composition of most abundant cultured plant species-specific bacteria. The abundance of Sphingomonas was significantly reduced. Sphingomonas isolates from warmed plots represented different phylotypes, had different physiological traits and were better adapted to higher temperatures. Among Methylobacterium isolates, a novel phylotype with a specific mxaFtype was cultured from plants of warmed plots while the most abundant phylotype cultured from control plots was strongly reduced. This study clearly showed a correlation of long-term surface warming with changes in the plant physiology and the development of a physiologically and genetically adapted phyllosphere microbiota.

The insect antimicrobial peptide cecropin A disrupts uropathogenic Escherichia coli biofilms.

Current antibiotics cannot eradicate uropathogenic Escherichia coli (UPEC) biofilms, leading to recurrent urinary tract infections. Here, we show that the insect antimicrobial peptide cecropin A (CecA) can destroy planktonic and sessile biofilm-forming UPEC cells, either alone or when combined with the antibiotic nalidixic acid (NAL), synergistically clearing infection in vivo without off-target cytotoxicity. The multi-target mechanism of action involves outer membrane permeabilization followed by biofilm disruption triggered by the inhibition of efflux pump activity and interactions with extracellular and intracellular nucleic acids. These diverse targets ensure that resistance to the CecA + NAL combination emerges slowly. The antimicrobial mechanisms of CecA, thus, extend beyond pore-forming activity to include an unanticipated biofilm-eradication process, offering an alternative approach to combat antibiotic-resistant UPEC infections.

Dissection of heterocellular cross-talk in vascularized cardiac tissue mimetics.

Cellular specialization and interaction with other cell types in cardiac tissue is essential for the coordinated function of cell populations in the heart. The complex interplay between cardiomyocytes, endothelial cells and fibroblasts is necessary for adaptation but can also lead to pathophysiological remodeling. To understand this complex interplay, we developed 3D vascularized cardiac tissue mimetics (CTM) to study heterocellular cross-talk in hypertrophic, hypoxic and fibrogenic environments. This 3D platform responds to physiologic and pathologic stressors and mimics the microenvironment of diseased tissue. In combination with endothelial cell fluorescence reporters, these cardiac tissue mimetics can be used to precisely visualize and quantify cellular and functional responses upon stress stimulation. Utilizing this platform, we demonstrate that stimulation of α/ß-adrenergic receptors with phenylephrine (PE) promotes cardiomyocyte hypertrophy, metabolic maturation and vascularization of CTMs. Increased vascularization was promoted by conditioned medium of PE-stimulated cardiomyocytes and blocked by inhibiting VEGF or upon ß-adrenergic receptor antagonist treatment, demonstrating cardiomyocyte-endothelial cross-talk. Pathophysiological stressors such as severe hypoxia reduced angiogenic sprouting and increased cell death, while TGF ß2 stimulation increased collagen deposition concomitant to endothelial-to-mesenchymal transition. In sum, we have developed a cardiac 3D culture system that reflects native cardiac tissue function, metabolism and morphology - and for the first time enables the tracking and analysis of cardiac vascularization dynamics in physiology and pathology.

Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2.

The endocannabinoid (EC) system has been implicated in the pathogenesis of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). With the current study we aimed to verify the modulatory effect of endocannabinoid receptor 1 (CB1)-signaling on perilipin 2 (PLIN2)-mediated lipophagy. Here, we demonstrate that a global knockout of the cannabinoid receptor 1 gene (CB1-/-) reduced the expression of the lipid droplet binding protein PLIN2 in the livers of CB1-/- and hepatitis B surface protein (HBs)-transgenic mice, which spontaneously develop hepatic steatosis. In addition, the pharmacologic activation and antagonization of CB1 in cell culture also caused an induction or reduction of PLIN2, respectively. The decreased PLIN2 expression was associated with suppressed lipogenesis and triglyceride (TG) synthesis and enhanced autophagy as shown by increased colocalization of LC3B with lysosomal-associated membrane protein 1 (LAMP1) in HBs/CB1-/- mice. The induction of autophagy was further supported by the increased expression of LAMP1 in CB1-/- and HBs/CB1-/- mice. LAMP1 and PLIN2 were co-localized in HBs/CB1-/- indicating autophagy of cytoplasmic lipid droplets (LDs) i.e., lipophagy. Lipolysis of lipid droplets was additionally indicated by elevated expression of lysosomal acid lipase. In conclusion, these results suggest that loss of CB1 signaling leads to reduced PLIN2 abundance, which triggers lipophagy. Our new findings about the association between CB1 signaling and PLIN2 may stimulate translational studies analyzing new diagnostic and therapeutic options for NAFLD.

Mitochondrial Oxidative Stress Impairs Energy Metabolism and Reduces Stress Resistance and Longevity of C. elegans.

INTRODUCTION: Mitochondria supply cellular energy and are key regulators of intrinsic cell death and consequently affect longevity. The nematode Caenorhabditis elegans is frequently used for lifespan assays. Using paraquat (PQ) as a generator of reactive oxygen species, we here describe its effects on the acceleration of aging and the associated dysfunctions at the level of mitochondria. METHODS: Nematodes were incubated with various concentrations of paraquat in a heat-stress resistance assay (37°C) using nucleic staining. The most effective concentration was validated under physiological conditions, and chemotaxis was assayed. Mitochondrial membrane potential (ΔΨm) was measured using rhodamine 123, and activity of respiratory chain complexes determined using a Clark-type electrode in isolated mitochondria. Energetic metabolites in the form of pyruvate, lactate, and ATP were determined using commercial kits. Mitochondrial integrity and structure was investigated using transmission electron microscopy. Live imaging after staining with fluorescent dyes was used to measure mitochondrial and cytosolic ROS. Expression of longevity- and mitogenesis-related genes were evaluated using qRT-PCR. RESULTS: PQ (5 mM) significantly increased ROS formation in nematodes and reduced the chemotaxis, the physiological lifespan, and the survival in assays for heat-stress resistance. The number of fragmented mitochondria significantly increased. The ∆Ψm, the activities of complexes I-IV of the mitochondrial respiratory chain, and the levels of pyruvate and lactate were significantly reduced, whereas ATP production was not affected. Transcript levels of genetic marker genes, atfs-1, atp-2, skn-1, and sir-2.1, were significantly upregulated after PQ incubation, which implicates a close connection between mitochondrial dysfunction and oxidative stress response. Expression levels of aak-2 and daf-16 were unchanged. CONCLUSION: Using paraquat as a stressor, we here describe the association of oxidative stress, restricted energy metabolism, and reduced stress resistance and longevity in the nematode Caenorhabditis elegans making it a readily accessible in vivo model for mitochondrial dysfunction.

Insects in anthelminthics research: Lady beetle-derived harmonine affects survival, reproduction and stem cell proliferation of Schistosoma mansoni.

Natural products have moved into the spotlight as possible sources for new drugs in the treatment of helminth infections including schistosomiasis. Surprisingly, insect-derived compounds have largely been neglected so far in the search for novel anthelminthics, despite the generally recognized high potential of insect biotechnology for drug discovery. This motivated us to assess the antischistosomal capacity of harmonine, an antimicrobial alkaloid from the harlequin ladybird Harmonia axyridis that raised high interest in insect biotechnology in recent years. We observed remarkably pleiotropic effects of harmonine on physiological, cellular, and molecular processes in adult male and female Schistosoma mansoni at concentrations as low as 5 µM in vitro. This included tegumental damage, gut dilatation, dysplasia of gonads, a complete stop of egg production at 10 µM, and increased production of abnormally shaped eggs at 5 µM. Motility was reduced with an EC50 of 8.8 µM and lethal effects occurred at 10-20 µM within 3 days of culture. Enzyme inhibition assays revealed acetylcholinesterase (AChE) as one potential target of harmonine. To assess possible effects on stem cells, which represent attractive anthelminthic targets, we developed a novel in silico 3D reconstruction of gonads based on confocal laser scanning microscopy of worms after EdU incorporation to allow for quantification of proliferating stem cells per organ. Harmonine significantly reduced the number of proliferating stem cells in testes, ovaries, and also the number of proliferating parenchymal neoblasts. This was further supported by a downregulated expression of the stem cell markers nanos-1 and nanos-2 in harmonine-treated worms revealed by quantitative real-time PCR. Our data demonstrate a multifaceted antischistosomal activity of the lady beetle-derived compound harmonine, and suggest AChE and stem cell genes as possible targets. Harmonine is the first animal-derived alkaloid detected to have antischistosomal capacity. This study highlights the potential of exploiting insects as a source for the discovery of anthelminthics.

Parendozoicomonas haliclonae gen. nov. sp. nov. isolated from a marine sponge of the genus Haliclona and description of the family Endozoicomonadaceae fam. nov. comprising the genera Endozoicomonas, Parendozoicomonas, and Kistimonas.

Two Gram-stain-negative, facultative anaerobic, motile, rod-shaped strains, S-B4-1UT and JOB-63a, forming small whitish transparent colonies on marine agar, were isolated from a sponge of the genus Haliclona. The strains shared 99.7% 16S rRNA gene sequence identity and a DNA-DNA hybridization value of 100%, but were differentiated by genomic fingerprinting using rep-PCRs. 16S rRNA gene sequence phylogeny placed the strains as a sister branch to the monophyletic genus Endozoicomonas (Oceanospirillales; Gammaproteobacteria) with 92.3-94.3% 16S rRNA gene sequence similarity to Endozoicomonas spp., 91.9 and 92.1% to Candidatus Endonucleobacter bathymodiolin, and 91.9 to 92.1% to the type strains of Kistimonas spp. Core genome based phylogeny of strain S-B4-1UT confirmed the phylogenetic placement. Major fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c) and 8 (C18:1 ω7c/C18:1 ω6c) followed by C10:0 3-OH, C16:0, and C18:0. The G+C content was 50.1-51.4mol%. The peptidoglycan diamino acid of strain S-B4-1UT was meso-diaminopimelic acid, the predominant polyamine spermidine, the major respiratory quinone ubiquinone Q-9; phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine were major polar lipids. Based on the clear phylogenetic distinction, the genus Parendozoicomonas gen. nov. is proposed, with Parendozoicomonas haliclonae sp. nov. as type species and strain S-B4-1UT (=CCM 8713T=DSM 103671T=LMG 29769T) as type strain and JOB-63a as a second strain of the species. Based on the 16S rRNA gene sequence phylogeny of the Oceanospirillales within the Gammaproteobacteria, the Endozoicomonaceae fam. nov. is proposed including the genera Endozoicomonas, Parendozoicomonas, and Kistimonas as well as the Candidatus genus Endonucleobacter.

Proposal of Litorimonas haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A bright-orange-pigmented, Gram-stain-negative, motile, and rod-shaped bacterium, strain MAA42T, was isolated from a marine sponge of the genus Haliclona, which is in long-time culture in a marine aquarium system at the Justus Liebig University Giessen, Germany. The strain grew at 4-34 °C (optimum 28 °C), in the presence of 0.5-9.5 % (w/v) NaCl (optimum 3.5 %) and at pH 4.5-10.0 (optimum pH 7.5). Strain MAA42T shared the highest 16S rRNA gene sequence similarity (98.1 %) with the type strain of Litorimonas taeanensis. Sequence similarities to all other closely related type strains were below 97 %. DNA-DNA hybridization of strain MAA42T with L. taeanensis DSM 22008T resulted in values of 4.7 % (reciprocal 17.7 %). Major cellular fatty acids of strain MAA42T were C18 : 1ω7c (66.2 %), C18 : 1 2-OH (17.4 %), and C18 : 0 (14.1 %). Spermidine was predominant in the polyamine pattern, and ubiquinone Q-10 was the major respiratory quinone. The polar lipid profile contained the major compounds phosphatidylglycerol, monoglycosyldiglyceride, three unidentified phospholipids, and one unidentified glycolipid. Glucuronopyranosyldiglyceride was present as a minor compound. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The genomic DNA G+C content was 52.8 mol%. Based on the genotypic, chemotaxonomic, and phenotypic analyses, strain MAA42T represents a novel species of the genus Litorimonas, for which the name Litorimonas haliclonae is proposed. The type strain is MAA42T (=CCM 8709T=CIP 111178T=LMG 29765T).

Selective release of circRNAs in platelet-derived extracellular vesicles.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell-type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density-gradient centrifugation, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.

Development of a synthetic tissue engineered three-dimensional printed bioceramic-based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro.

Over the last decade there have been increasing efforts to develop three-dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica-containing calcium alkali orthophosphate, using, first, a replica technique - the Schwartzwalder-Somers method - and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 109 cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D-printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 109 cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the sn-2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLA2α inhibitor.

Winogradskyella haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A yellow-pigmented, Gram-stain-negative, motile and rod-shaped bacterium, strain M1A16T, was isolated from the internal tissue of a sponge of the genus Haliclona, which was long-term cultured in the CEMarin aquaria system at Justus Liebig University of Giessen. The strain grew well at 20-32 °C (optimum 25 °C), in the presence of 0-6 % NaCl (optimum 3 %), and at pH 5.5-9.0 (optimum pH 7.0-8.0). Phylogenetic analysis based on its 16S rRNA gene sequence placed the strain within the monophyletic cluster of the genus Winogradskyella with highest sequence similarity to Winogradskyella jejuensis CP32T (98.3 % 16S rRNA gene sequence similarity). Sequence similarities to all other type strains were 98.0 % or less. DNA-DNA hybridization of strain M1A16T with W. jejuensis CP32T resulted in hybridization values of 44.1 % (reciprocal 68.1 %). Major cellular fatty acids of strain M1A16T were iso-C15 : 1 G (18.1 %), iso-C15 : 0 (13.7 %), C16 : 1ω7c (12.9 %), iso-C17 : 0 3-OH (10.6 %) and iso-C16 : 0 3-OH (10.2 %). The overall polyamine content was very low with major components being cadaverine, spermidine and sym-homospermidine. The major quinone was menaquinone MK-6. The polar lipid profile contained predominantly phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids devoid of a detectable functional group. The genomic DNA G+C content was 32.7 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic analyses, strain M1A16T represents a novel species of the genus Winogradskyella, for which the name Winogradskyella haliclonae sp. nov. is proposed. The type strain is M1A16T (=DSM 103138T=CCM 8681T=LMG 29588T=CIP 111091T).

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells.

Many ribonucleases (RNases) are considered as promising tools for antitumor therapy because of their selective cytotoxicity toward cancer cells. Binase, the RNase from Bacillus pumilus, triggers apoptotic response in cancer cells expressing RAS oncogene which is mutated in a large percentage of prevalent and deadly malignancies including colorectal cancer. The specific antitumor effect of binase toward RAS-transformed cells is due to its direct binding of RAS protein and inhibition of downstream signaling. However, the delivery of proteins to the intestine is complicated by their degradation in the digestive tract and subsequent loss of therapeutic activity. Therefore, the search of new systems for effective delivery of therapeutic proteins is an actual task. This study is aimed to the investigation of antitumor effect of binase immobilized on natural halloysite nanotubes (HNTs). Here, we have developed the method of binase immobilization on HNTs and optimized the conditions for the enzyme loading and release (i); we have found the non-toxic concentration of pure HNTs which allows to distinguish HNTs- and binase-induced cytotoxic effects (ii); using dark-field and fluorescent microscopy we have proved the absorption of binase-loaded HNTs on the cell surface (iii) and demonstrated that binase-halloysite nanoformulations possessed twice enhanced cytotoxicity toward tumor colon cells as compared to the cytotoxicity of binase itself (iv). The enhanced antitumor activity of biocompatible binase-HNTs complex confirms the advisability of its future development for clinical practice.

Non-pathogenic Rhizobium radiobacter F4 deploys plant beneficial activity independent of its host Piriformospora indica.

The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) was originally characterized as an endofungal bacterium in the beneficial endophytic Sebacinalean fungus Piriformospora indica. Although attempts to cure P. indica from RrF4 repeatedly failed, the bacterium can easily be grown in pure culture. Here, we report on RrF4's genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic R. radiobacter (formerly: Agrobacterium tumefaciens) C58, except vibrant differences in both the tumor-inducing (pTi) and the accessor (pAt) plasmids, which can explain the loss of RrF4's pathogenicity. Similar to its fungal host, RrF4 colonizes plant roots without host preference and forms aggregates of attached cells and dense biofilms at the root surface of maturation zones. RrF4-colonized plants show increased biomass and enhanced resistance against bacterial leaf pathogens. Mutational analysis showed that, similar to P. indica, resistance mediated by RrF4 was dependent on the plant's jasmonate-based induced systemic resistance (ISR) pathway. Consistent with this, RrF4- and P. indica-induced pattern of defense gene expression were similar. In clear contrast to P. indica, but similar to plant growth-promoting rhizobacteria, RrF4 colonized not only the root outer cortex but also spread beyond the endodermis into the stele. On the basis of our findings, RrF4 is an efficient plant growth-promoting bacterium.

Isolation, enrichment and primary characterisation of vitelline cells from Schistosoma mansoni obtained by the organ isolation method.

In the emerging era of post-genomic research on schistosomes, new methods are required to functionally analyse genes of interest in more detail. Among other tools, schistosome cell lines are needed to overcome present research constraints. Based on a recently established organ isolation protocol for adult Schistosoma mansoni, we report here on the successful enrichment of vitellarium tissue and isolation of vitelline cells. Morphological analyses performed by bright field, fluorescence, scanning and transmission electron microscopy showed typical features of S1 to S4 stage vitelline cells. In addition, molecular analyses using reverse transcription-PCR confirmed the identity of vitelline cells. Cytological and physiological studies included staining experiments with viability dyes and a neutral lipid stain, as well as calcium (Ca2+) imaging. Together they demonstrated cell viability, the possibility to define the differentiation stage of individual vitelline cells, and the suitability to investigate Ca(2+)-associated processes herein. Finally, fluorescence-activated cell sorting was shown to be a convenient way to separate and enrich S1 to S4 stage vitelline cells. In summary, these results demonstrate the expedience of the organ isolation protocol to obtain vitellarium tissue. Importantly, the protocol allows vitelline cells representing defined differentiation stages to be purified, which can be cultured in vitro and used to investigate diverse aspects of schistosome reproductive biology in the post-genomic era.

Influenza virus-induced caspase-dependent enlargement of nuclear pores promotes nuclear export of viral ribonucleoprotein complexes.

UNLABELLED: Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection. IMPORTANCE: In contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The report provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.