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.

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.

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.

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).

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).

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.

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.

Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

Hepatitis B virus infection is dependent on cholesterol in the viral envelope.

The viral and cellular determinants leading to binding and entry of hepatitis B virus (HBV) are still not fully understood. We found that HBV infection of primary hepatocyte cultures is dependent on the presence of cholesterol in the viral envelope. Extraction of cholesterol from HBV purified from plasma of HBV-infected patients with methyl-beta-cyclodextrin (MbetaCD) leads to a strongly reduced level of infection. The cholesterol-depleted virions showed higher buoyant density (1.23 versus 1.17 g ml(-1)), a smaller diameter (39 versus 48 nm), but maintained particle integrity, antigenicity and ability to bind to hepatocytes. Although addition of exogenous cholesterol and cholesterol analogues restored the physical appearance of cholesterol-depleted virions, infectivity was only regained by cholesterol add-back. Infectivity of HBV produced from cell culture in the presence of inhibitors of cholesterol-synthesis is severely impaired. Interestingly, cholesterol extraction from cellular membranes, incubation with filipin and the protein tyrosine kinase inhibitor genistein showed no effect on HBV infection, excluding a role of lipid rafts for the infection process of HBV. In summary, presence of cholesterol within the viral envelope is not important for viral binding, but indispensable for the entry process of HBV and might be important for a later step in viral uptake, e.g. fusion in a yet unknown compartment.

Mechanism of action of tin-containing fluoride solutions as anti-erosive agents in dentine - an in vitro tin-uptake, tissue loss, and scanning electron microscopy study.

Solutions containing tin and fluoride exhibit remarkable anti-erosive properties with tin ions as a major agent. To elucidate its mechanism of action in dentine, the tin uptake on and in the tissue was investigated and related to histological findings and substance loss. Samples were treated twice daily, each treatment lasting for 2 min, with fluoride solutions [pH 4.5; 1,500 parts per million (p.p.m.) F] containing 2,100, 1,400, or 400 p.p.m. Sn as SnCl(2). In experiments 1 and 2, samples were eroded with citric acid (pH 2.3) six times each day, each treatment lasting for 5 min; in experiment 2, the demineralized organic matrix was continuously digested by collagenase; in experiment 3, no erosive challenges were performed. Sample surfaces and cross-sections were investigated using energy dispersive X-ray spectroscopy, scanning electron microscopy, and profilometry. Surface retention of tin was found in almost all treatment groups and was highest in experiment 2. On cross-sections, tin was retained within the organic matrix; in mineralized areas, tin was found mainly within a depth of 10 mum. Test solutions inhibited substance loss significantly; in experiment 2, the effect was dose-dependent. Erosion inhibition seemed to depend mainly on the incorporation of tin in the mineralized dentine when the organic portion was preserved, but on surface precipitation when the organic portion was continuously digested.

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 %.

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.

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.

Effects of toothbrushing force on the mineral content and demineralized organic matrix of eroded dentine.

Eroded dentine has a complex histological structure, and its organic fraction becomes increasingly exposed as a result of the continual action of acids. The present study sought to investigate the effects of brushing forces up to 4 N on mineralized and organic dentine fractions. The study was a cyclic demineralization and remineralization experiment (carried out over 9 d). Erosion was performed with HCl (6 x 2 min d-1), pH 1.6. Samples exposed to erosion alone served as controls; test samples were eroded and brushed with a powered toothbrush (2 x 15 s d-1), applying forces of 2, 3, or 4 N. Samples were analysed (using profilometry and longitudinal microradiography) before and after the removal of superficial organic material with collagenase. Randomly selected samples were subjected to scanning electron microscopy. Demineralized organic material was present on all samples regardless of brushing force. Loss values (determined using profilometry) after erosion only, and after brushing with forces of 2, 3, and 4 N, were 11.7 +/- 5.1, 13.6 +/- 11.2, 30.7 +/- 19.0, and 25.5 +/- 20.3, respectively, before treatment with collagenase, and 111.7 +/- 11.6, 122.0 +/- 11.8, 121.9 +/- 15.7, and 123.0 +/- 12.0, respectively, after treatment with collagenase. Microradiography confirmed the results. Significant effects of brushing force were only found on the demineralized organic fraction, and mineral loss was unaffected. The notion that eroded dentine is particularly prone to abrasion should be reconsidered.

Tin-containing fluoride solutions as anti-erosive agents in enamel: an in vitro tin-uptake, tissue-loss, and scanning electron micrograph study.

Tin-containing fluoride solutions can reduce erosive tissue loss, but the effects of the reaction between tin and enamel are still not clear. During a 10-d period, enamel specimens were cyclically demineralized (0.05 M citric acid, pH 2.3, 6 x 5 min d(-1)) and remineralized (between the demineralization cycles and overnight). In the negative-control group, no further treatment was performed. Three groups were treated (2 x 2 min d(-1)) with tin-containing fluoride solutions (400, 1,400 or 2,100 ppm Sn2+, all 1,500 ppm F-, pH 4.5). Three additional groups were treated with test solutions twice daily, but without demineralization. Tissue loss was determined profilometrically. Energy-dispersive X-ray spectroscopy was used to measure the tin content on and within three layers (10 mum each) beneath the surface. In addition, scanning electron microscopy was conducted. All test preparations significantly reduced tissue loss. Deposition of tin on surfaces was higher without erosion than with erosion, but no incorporation of tin into enamel was found without demineralization. Under erosive conditions, both highly concentrated solutions led to the incorporation of tin up to a depth of 20 mum; the less-concentrated solution led to small amounts of tin in the outer 10 mum. The efficacy of tin-containing solutions seems to depend mainly on the incorporation of tin into enamel.

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.

PMN-mediated immune reactions against Eimeria bovis.

For successful in vivo infection, Eimeria bovis sporozoites have to traverse the mucosal layer of the ileum to infect lymphatic endothelial cells and may, thereby, be exposed to the interstitial fluid and to the lymph representing potential targets for leukocytes. To mimic this situation in vitro, we exposed E. bovis sporozoites to bovine PMN and found enhanced elimination of the parasites. Addition of immune serum clearly increased these reactions, whereas neonatal calf serum had no effect, thus proposing a PMN-derived antibody-dependent cytotoxicity. Scanning and transmission electron microscopy showed PMN engulfing sporozoites or extending filopodia towards them and occasionally incorporating the parasites. PMN reacted with enhanced transcription of IL-6, MCP-1, GROalpha, TNF-alpha, and iNOS genes after exposure to sporozoites while stimulation with merozoite-antigen, in addition, upregulated IL-8, IP-10 and IL-12 gene transcription. Furthermore, enhanced in vitro oxidative burst and phagocytic activities were observed after contact of PMN with viable sporozoites. To verify the potential role of PMN in the in vivo situation, we analysed the general phagocytic and oxidative burst activities of PMN obtained ex vivo from E. bovis experimentally infected calves. Enhanced levels of both activities were found early p.i. (1-5 days) and towards the end of the first schizogony (days 13-22 p.i.) underlining the in vitro data. Our results suggest that PMN-mediated, innate immune reactions play an important role in the early immune response to E. bovis infections in calves.