Seed Architecture Shapes Embryo Metabolism in Oilseed Rape

Constrained to develop within the seed, the plant embryo must adapt its shape and size to fit the space available. Here, we demonstrate how this adjustment shapes metabolism of photosynthetic embryo. Noninvasive NMR-based imaging of the developing oilseed rape (Brassica napus) seed illustrates that, following embryo bending, gradients in lipid concentration became established. These were correlated with the local photosynthetic electron transport rate and the accumulation of storage products. Experimentally induced changes in embryo morphology and/or light supply altered these gradients and were accompanied by alterations in both proteome and metabolome. Tissue-specific metabolic models predicted that the outer cotyledon and hypocotyl/radicle generate the bulk of plastidic reductant/ATP via photosynthesis, while the inner cotyledon, being enclosed by the outer cotyledon, is forced to grow essentially heterotrophically. Under field-relevant high-light conditions, major contribution of the ribulose-1,5-bisphosphate carboxylase/oxygenase–bypass to seed storage metabolism is predicted for the outer cotyledon and the hypocotyl/radicle only. Differences between in vitro– versus in planta–grown embryos suggest that metabolic heterogeneity of embryo is not observable by in vitro approaches. We conclude that in vivo metabolic fluxes are locally regulated and connected to seed architecture, driving the embryo toward an efficient use of available light and space.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

Community structure of soil oribatida (acari) two years after windthrow in the high tatra mountains

In November 2004 a catastrophic windstorm destroyed a large part of the spruce forest in the Tatra National Park (Slovakia). The majority of the windthrown area was cleared; only a small part was left uncleared, thereby allowing regeneration by natural succession. The aim of the present study was to assess the impact of the different forestry practices on soil Oribatida. Three different stands were selected for the study, where sampling took place in June and October 2006: control forest stands (REF), windthrown stands left for natural development (NEX) and clear-cut windthrown stands (EXT). The mean abundance of Oribatida recorded in REF and NEX stands was significantly higher than in EXT stands. Kruskal-Wallis test of mean abundance of adults as well as juveniles confirmed significant influence of treatment and date. The highest abundance of adults was found in control forest stands (REF). Post hoc multiple comparison proved significantly lower abundance of adults in clear-cut stands (EXT) compared with REF. The mean abundance of adults and juveniles was several times higher in stands left for natural development (NEX) than in EXT stands. The highest species richness was observed in REF, followed by NEX and EXT stands. Ordination method showed differences in species composition between studied treatments. Furthermore, a much lower abundance of Hermannia gibba, a dweller of leaf litter and upper soil layers, was recorded in cleared stands compared to the other stands. Indeed, windthrown stands had an obvious lower species richness than control stands. The ordination method used demonstrated a significant influence of both treatment and sampling date on the abundance and species richness of Oribatida. The present study showed that clear-cutting of wind-damaged spruce forest markedly decreases the abundance of soil Oribatida compared with windthrown forest stands left to natural succession.     

Effects of Sphingomyelin Headgroup Size on Interactions with Ceramide

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide’s aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals’ forces between saturated chains of PCer and SM analogs.     

A Membrane-Translocating Peptide Penetrates into Bilayers without Significant Bilayer Perturbations

Using a high throughput screen, we have identified a family of 12-residue long peptides that spontaneously translocate across membranes. These peptides function by a poorly understood mechanism that is very different from that of the well-known, highly cationic cell penetrating peptides such as the tat peptide from HIV. The newly discovered translocating peptides can carry polar cargoes across synthetic bilayers and across cellular membranes quickly and spontaneously without disrupting the membrane. Here we report on the biophysical characterization of a representative translocating peptide from the selected family, TP2, as well as a negative control peptide, ONEG, from the same library. We measured the binding of the two peptides to lipid bilayers, their secondary structure propensities, their dispositions in bilayers by neutron diffraction, and the response of the bilayer to the peptides. Compared to the negative control, TP2 has a greater propensity for membrane partitioning, although it still binds only weakly, and a higher propensity for secondary structure. Perhaps most revealing, TP2 has the ability to penetrate deep into the bilayer without causing significant bilayer perturbations, a property that may help explain its ability to translocate without bilayer permeabilization.     

Improved recellularization of ex vivo vascular scaffolds using directed transport gradients to modulate ECM remodeling

The regeneration of functional, clinically viable, tissues from acellular ex vivo tissues has been problematic largely due to poor nutrient transport conditions that limit cell migration and integration. Compounding these issues are subcellular pore sizes that necessarily requires extracellular matrix (ECM) remodeling in order for cells to migrate and regenerate the tissue. The aim of the present work was to create a directed growth environment that allows cells to fully populate an ex vivo‐derived vascular scaffold and maintain viability over extended periods. Three different culture conditions using single (one nutrient source) or dual perfusion bioreactor systems (two nutrients sources) were designed to assess the effect of pressure and nutrient gradients under either low (50/30 mmHg) or high (120/80) relative pressure conditions. Human myofibroblasts were seeded to the ablumenal periphery of an ex vivo‐derived vascular scaffold using a collagen/hydrogel cell delivery system. After 30 days culture, total cell density was consistent between groups; however, significant variation was noted in cell distribution and construct mechanics as a result of differing perfusion conditions. The most aggressive transport gradient was developed by the single perfusion low‐pressure circuits and resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source). These investigations illustrate the influence of directed nutrient gradients where precisely controlled perfusion conditions significantly affects cell migration, distribution and function, resulting in pronounced effects on construct mechanics during early remodeling events. Biotechnol. Bioeng. 2013; 110: 2035–2045. © 2013 Wiley Periodicals, Inc.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

HLA-C, DRB1 and DQB1 alleles involved in genetic predisposition to psoriasis vulgaris in the Slovak population

Psoriasis vulgaris is a complex chronic skin disease with immunological and genetic background. The most important predisposing genetic factors in psoriasis are genes of the human leukocyte antigen (HLA) region. Accumulative evidence has shown that several HLA alleles are closely associated with psoriasis; however, they tend to vary in different racial and ethnic backgrounds. One hundred forty-seven unrelated Slovak patients with psoriasis vulgaris (average age at onset 28?±?14 years) were genotyped for the HLA-C, DQB1 and DRB1 alleles by the polymerase chain reaction using sequence-specific primers. Allele frequencies observed in the group of psoriatic patients were compared to those obtained in the ethnically matched control group comprising 194 subjects with no history of psoriasis. Susceptibility to psoriasis vulgaris in our study group is significantly associated with HLA-C*06 (odds ratio (OR)?=?3.85), DRB1*07 (OR?=?2.56) and DQB1*02 (OR?=?1.09), respectively, whereas DRB*01 (OR?=?0.05) is associated negatively. Hereby, we provide the first report on the association of HLA-C, DRB1 and DQB1 alleles with psoriasis in the Slovak population. Our findings confirm HLA-C*06 and DRB1*07 as the most important genetic risk factors for psoriasis. However, the role of HLA genes as causative in the pathogenesis of the disease remains unclear. Identification of genetic factors that increase the risk of psoriasis is a precondition that helps to elucidate the pathogenesis of this troubling disease and identify targets for a more specific and effective therapy.     

Comparison of different treatments for LM and SEM studies and systematic value of pollen grains in Cyperaceae

The pollen grain morphology of 30 species of 27 genera from the four subfamilies of the Cyperaceae have been studied with LM and SEM. Several methods were tested in order to find the optimal treatment for the delicate Cyperaceae pollen grains. For LM, treatment with wetting agent Agepon and KOH yielded the best results, while critical point drying (CPD) after fixation with alcohol proved to be the best method for studying pollen grains of Cyperaceae with SEM. Our results show that the Mapanioideae have a pollen grain type (only one distal ulcus) clearly different from the other Cyperaceae. Representatives of the examined Sclerioideae and Caricoideae show a similar pollen grain type (mostly one distal ulcus and three lateral pores) while in the Cyperoideae different pollen grain types are found.