Comparative Metabolomic Analysis of Four Fabaceae and Relationship to In Vitro Nematicidal Activity against Xiphinema index.

The grapevine fanleaf virus (GFLV), responsible for fanleaf degeneration, is spread in vineyards by the soil nematode Xiphinema index. Nematicide molecules were used to limit the spread of the disease until they were banned due to negative environmental impacts. Therefore, there is a growing interest in alternative methods, including plant-derived products with antagonistic effects to X. index. In this work, we evaluated the nematicidal potential of the aerial parts and roots of four Fabaceae: sainfoin (Onobrychis viciifolia), birdsfoot trefoil (Lotus corniculatus), sweet clover (Melilotus albus), and red clover (Trifolium pratense), as well as that of sainfoin-based commercial pellets. For all tested plants, either aerial or root parts, or both of them, exhibited a nematicidal effect on X. index in vitro, pellets being as effective as freshly harvested plants. Comparative metabolomic analyses did not reveal molecules or molecule families specifically associated with antagonistic properties toward X. index, suggesting that the nematicidal effect is the result of a combination of different molecules rather than associated with a single compound. Finally, scanning electron microscope observations did not reveal the visible impact of O. viciifolia extract on X. index cuticle, suggesting that alteration of the cuticle may not be the primary cause of their nematicidal effect.

Developmental pattern of grapevine (Vitis vinifera L.) berry cuticular wax: Differentiation between epicuticular crystals and underlying wax.

The grapevine berry surface is covered by a cuticle consisting of cutin and various lipophilic wax compounds. The latter build the main barrier for transpirational water loss and protect the fruit against environmental factors e.g. pests, mechanical impacts or radiation. The integrety of the fruit surface is one important key factor for post-harvest quality and storage of fruits. Nonetheless, the developmental pattern of cuticular wax was so far only investigated for a very limited number of fruits. Therefore, we performed comparative investigations on the compositional and morphological nature of epicuticular wax crystals and underlying wax during fruit development in Vitis vinifera. The main compound oleanolic acid belongs to the pentacyclic triterpenoids, which occur very early in the development in high amounts inside the cuticle. The amount increases until veraison and decreases further during ripening. In general, very-long chain aliphatic (VLCA) compounds are present in much smaller amounts and alcohols and aldehydes follow the same trend during development. In contrast, the amount of fatty acids constantly increases from fruit set to ripening while wax esters only occur in significant amount at veraison and increase further. Wax crystals at the fruit surface are solely composed of VLCAs and the morphology changes during development according to the compositional changes of the VLCA wax compounds. The remarkable compositional differences between epicuticular wax crystals and the underlying wax are important to understand in terms of studying grape-pest interactions or the influence of environmental factors, since only wax crystals directly face the environment.

Towards Sensor-Based Phenotyping of Physical Barriers of Grapes to Improve Resilience to Botrytis Bunch Rot.

Botrytis bunch rot is one of the economically most important fungal diseases in viticulture (aside from powdery mildew and downy mildew). So far, no active defense mechanisms and resistance loci against the necrotrophic pathogen are known. Since long, breeders are mostly selecting phenotypically for loose grape bunches, which is recently the most evident trait to decrease the infection risk of Botrytis bunch rot. This study focused on plant phenomics of multiple traits by applying fast sensor technologies to measure berry impedance (Z REL ), berry texture, and 3D bunch architecture. As references, microscopic determined cuticle thickness (MS CT ) and infestation of grapes with Botrytis bunch rot were used. Z REL hereby is correlated to grape bunch density OIV204 (r = -0.6), cuticle thickness of berries (r = 0.61), mean berry diameter (r = -0.63), and Botrytis bunch rot (r = -0.7). However, no correlation between Z REL and berry maturity or berry texture was observed. In comparison to the category of traditional varieties (mostly susceptible), elite breeding lines show an impressive increased Z REL value (+317) and a 1-µm thicker berry cuticle. Quantitative trait loci (QTLs) on LGs 2, 6, 11, 15, and 16 were identified for Z REL and berry texture explaining a phenotypic variance of between 3 and 10.9%. These QTLs providing a starting point for the development of molecular markers. Modeling of Z REL and berry texture to predict Botrytis bunch rot resilience revealed McFadden R 2 = 0.99. Taken together, this study shows that in addition to loose grape bunch architecture, berry diameter, Z REL , and berry texture values are probably additional parameters that could be used to identify and select Botrytis-resilient wine grape varieties. Furthermore, grapevine breeding will benefit from these reliable methodologies permitting high-throughput screening for additional resilience traits of mechanical and physical barriers to Botrytis bunch rot. The findings might also be applicable to table grapes and other fruit crops like tomato or blueberry.

Ancestral chemotypes of cultivated grapevine with resistance to Botryosphaeriaceae-related dieback allocate metabolism towards bioactive stilbenes.

Grapevine trunk diseases have devastating consequences on vineyards worldwide. European wild grapevines (Vitis vinifera subs. sylvestris) from the last viable population in Germany along the Rhine river showed variable degrees of resistance against Neofusicoccum parvum (strain Bt-67), a fungus associated with Botryosphaeriaceae-related dieback. Representative genotypes from different subclades of this population were mapped with respect to their ability to induce wood necrosis, as well as their defence responses in a controlled inoculation system. The difference in colonization patterns could be confirmed by cryo-scanning electron microscopy, while there was no relationship between vessel diameter and infection success. Resistant lines accumulated more stilbenes, that were in addition significantly partitioned to nonglycosylated viniferin trimers. By contrast, the susceptible genotypes accumulated less stilbenes with a significantly higher proportion of glycosylated piceid. We suggest a model in which in the resistant genotypes phenylpropanoid metabolism is channelled rapidly and specifically to the bioactive stilbenes. Our study specifies a resistant chemotype against grapevines trunk diseases and paves a way to breed for resistance against grapevine Botryosphaeriaceae-related dieback.

A perfusion bioreactor-based 3D model of the subarachnoid space based on a meningeal tissue construct.

BACKGROUND: Altered flow of cerebrospinal fluid (CSF) within the subarachnoid space (SAS) is connected to brain, but also optic nerve degenerative diseases. To overcome the lack of suitable in vitro models that faithfully recapitulate the intricate three-dimensional architecture, complex cellular interactions, and fluid dynamics within the SAS, we have developed a perfusion bioreactor-based 3D in vitro model using primary human meningothelial cells (MECs) to generate meningeal tissue constructs. We ultimately employed this model to evaluate the impact of impaired CSF flow as evidenced during optic nerve compartment syndrome on the transcriptomic landscape of MECs. METHODS: Primary human meningothelial cells (phMECs) were seeded and cultured on collagen scaffolds in a perfusion bioreactor to generate engineered meningeal tissue constructs. Engineered constructs were compared to human SAS and assessed for specific cell-cell interaction markers as well as for extracellular matrix proteins found in human meninges. Using the established model, meningeal tissue constructs were exposed to physiological and pathophysiological flow conditions simulating the impaired CSF flow associated with optic nerve compartment syndrome and RNA sequencing was performed. RESULTS: Engineered constructs displayed similar microarchitecture compared to human SAS with regards to pore size, geometry as well as interconnectivity. They stained positively for specific cell-cell interaction markers indicative of a functional meningeal tissue, as well as extracellular matrix proteins found in human meninges. Analysis by RNA sequencing revealed altered expression of genes associated with extracellular matrix remodeling, endo-lysosomal processing, and mitochondrial energy metabolism under pathophysiological flow conditions. CONCLUSIONS: Alterations of these biological processes may not only interfere with critical MEC functions impacting CSF and hence optic nerve homeostasis, but may likely alter SAS structure, thereby further impeding cerebrospinal fluid flow. Future studies based on the established 3D model will lead to new insights into the role of MECs in the pathogenesis of optic nerve but also brain degenerative diseases.

A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network.

Notch signaling plays a fundamental role in cellular differentiation and has been linked to human diseases, including cancer. We report the use of comprehensive RNAi analyses to dissect Notch regulation and its connections to cellular pathways. A cell-based RNAi screen identified 900 candidate Notch regulators on a genome-wide scale. The subsequent use of a library of transgenic Drosophila expressing RNAi constructs enabled large-scale in vivo validation and confirmed 333 of 501 tested genes as Notch regulators. Mapping the phenotypic attributes of our data on an interaction network identified another 68 relevant genes and revealed several modules of unexpected Notch regulatory activity. In particular, we note an intriguing relationship to pyruvate metabolism, which may be relevant to cancer. Our study reveals a hitherto unappreciated diversity of tissue-specific modulators impinging on Notch and opens new avenues for studying Notch regulation and function in development and disease.

Investigating native coronary artery endothelium in situ and in cell culture by scanning force microscopy.

The purpose of our studies is to better understand the morphology and functioning of the arteries and their changes in pathogenesis. The most frequently used imaging techniques are intravascular ultrasound, magnetic resonance imaging, and optical coherence tomography. These methods do not image cell-level structural details and only provide biomechanical properties indirectly. We present a new protocol for imaging the endothelial surface and measuring elastic properties of vascular tissue by scanning force microscopy. Full-thickness sections of native pig coronary arteries were prepared. In addition, cultured human umbilical vein endothelial cells were studied as an in vitro model system and for comparison. We encountered a variety of difficulties mostly due to the softness of vascular tissue which required significant adaptations of standard equipment: (i) a new specimen holder designed to stably immobilize the coronary arteries; (ii) a phase-contrast microscope incorporated for assessing the status of the cultured endothelial cells and positioning the scanning force microscope (SFM) tip at a site of interest; and (iii) a continuous exchange of the culture medium at 37 degrees C to assure viability of the cells in the SFM over extended times. We were thus able to investigate both fresh arterial tissue and living endothelial cells in a near-physiological environment. We present initial SFM images of vascular tissue at a spatial resolution similar to scanning electron microscopy, but which also provide a closer view of the bona fide structure of native tissue. Novel morphological features such as distinct granular particles were observed. Moreover, we report initial measurements of vascular tissue surface stiffness, obtained by indentation-type SFM.

Apoptosis and differentiation induced by staurosporine in granulosa tumor cells is coupled with activation of JNK and suppression of p38 MAPK.

We report here that staurosporine can induce apoptosis or differentiation of granulosa tumor cells depending on its dosage. In presence of staurosporine concentrations > 50 nM, apoptosis was triggered in human granulosa cell tumor cells COV434. In the presence of concentrations < 50 nM, the shape of the otherwise globular granulosa cells differentiated into a flattened epithelioid-like appearance. The process was associated by the induction of prostaglandin synthase-2 (PGS-2) and C/EBPbeta expression and by an increase in progesterone production in the supernatant culture medium. The observed effects of staurosporine were synergized by forskolin. With phosphorylation-specific Western blotting and protein kinase assays, it was demonstrated that staurosporine suppresses the phosphorylation of p38 and activates JNK. These results suggest that p38MAPK and JNK signal transduction pathways were involved in the regulation of granulosa cell differentiation by staurosporine. These results may indicate the usefulness of staurosporine or its analogs for the development of a future medical treatment of granulosa tumors.

The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence.

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.

Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs.

Calcium-dependent protein kinases play a pivotal role in calcium signalling in plants and some protozoa, including the malaria parasites. They are found in various subcellular locations, suggesting an involvement in multiple signal transduction pathways. Recently, Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) has been found in the membrane and organelle fraction of the parasite. The kinase contains three motifs for membrane binding at its N-terminus, a consensus sequence for myristoylation, a putative palmitoylation site and a basic motif. Endogenous PfCDPK1 and the in vitro translated kinase were both shown to be myristoylated. The supposed membrane attachment function of the basic cluster was experimentally verified and shown to participate together with N-myristoylation in membrane anchoring of the kinase. Using immunogold electron microscopy, the protein was detected in the parasitophorous vacuole and the tubovesicular system of the parasite. Mutagenesis of the predicted acylated residues and the basic motif confirmed that dual acylation and the basic cluster are required for correct targeting of Aequorea victoria green fluorescent protein to the parasitophorous vacuole, suggesting that PfCDPK1 as the leishmanial hydrophilic acylated surface protein B is a representative of a novel class of proteins whose export is dependent on a 'non-classical' pathway involving N-myristoylation/palmitoylation.