A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis

Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink–source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.     

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E ?) and with (E +) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E ? compared with E + fish (P < 0.0001); the estimated half-life of depletion in the E ? fish was 34 days, while in it was 55 days in the E + fish. To assess swimming behavior, zebrafish were monitored individually following a “startle-response” stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E ? fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E + fish. During the first minute following a tap stimulus (p < 0.05), E + fish swam twice as far as did E ? fish. In the E ? fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.     

A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.     

Plant response to climate change along the forest‐tundra ecotone in northeastern Siberia

Russia's boreal (taiga) biome will likely contract sharply and shift northward in response to 21st century climatic change, yet few studies have examined plant response to climatic variability along the northern margin. We quantified climate dynamics, trends in plant growth, and growth–climate relationships across the tundra shrublands and Cajander larch (Larix cajanderi Mayr.) woodlands of the Kolyma river basin (657 000 km²) in northeastern Siberia using satellite‐derived normalized difference vegetation indices (NDVI), tree ring‐width measurements, and climate data. Mean summer temperatures (T_s) increased 1.0 °C from 1938 to 2009, though there was no trend (P > 0.05) in growing year precipitation or climate moisture index (CMI_gy). Mean summer NDVI (NDVI_s) increased significantly from 1982 to 2010 across 20% of the watershed, primarily in cold, shrub‐dominated areas. NDVI_s positively correlated (P < 0.05) with T_s across 56% of the watershed (r = 0.52 ± 0.09, mean ± SD), principally in cold areas, and with CMI_gy across 9% of the watershed (r = 0.45 ± 0.06), largely in warm areas. Larch ring‐width measurements from nine sites revealed that year‐to‐year (i.e., high‐frequency) variation in growth positively correlated (P < 0.05) with June temperature (r = 0.40) and prior summer CMI (r = 0.40) from 1938 to 2007. An unexplained multi‐decadal (i.e., low‐frequency) decline in annual basal area increment (BAI) occurred following the mid‐20th century, but over the NDVI record there was no trend in mean BAI (P > 0.05), which significantly correlated with NDVI_s (r = 0.44, P < 0.05, 1982–2007). Both satellite and tree‐ring analyses indicated that plant growth was constrained by both low temperatures and limited moisture availability and, furthermore, that warming enhanced growth. Impacts of future climatic change on forests near treeline in Arctic Russia will likely be influenced by shifts in both temperature and moisture, which implies that projections of future forest distribution and productivity in this area should take into account the interactions of energy and moisture limitations.     

Scission of COPI and COPII Vesicles Is Independent of GTP Hydrolysis

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP‐binding proteins. Some reports describe that the scission of COP‐coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI‐ and COPII‐coated vesicles utilizing semi‐intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP‐PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.     

Spatial variability in structural and functional aspects of macrofauna communities and their environmental parameters in the Jade Bay (Wadden Sea Lower Saxony, southern North Sea)

Spatial distribution and functional structure of intertidal benthic macrofauna in relation to environmental variables in the Jade Bay (southern North Sea) were studied and compared with other intertidal areas of the Wadden Sea. A total of 128 stations covering the whole Jade Bay were sampled in summer 2009. A total of 114 taxa were found. Highest species numbers occurred in the subtidal areas, whereas highest mean abundances were found in the upper intertidal areas. Based on species abundance data, six significantly distinct macrofauna communities in the Jade Bay were identified and evaluated with multivariate statistics, univariate correlations and canonical correspondence analysis. Differences in these community patterns were caused by the response of the dominant species (Hydrobia ulvae, Tubificoides benedii, Pygospio elegans, Caulleriella killariensis, Scoloplos armiger, Urothoe poseidonis, Microprotopus maculatus) to prevailing environmental conditions along the gradient from the lower and exposed sandy intertidal areas via intermediate mixed sediments to the upper mudflat areas. Distribution patterns in relation to tidal zonation were best explained by variability in submergence time, Chlorophyll a (chl a) content and sediment composition (mud content), which are proxies for hydrodynamic conditions and food availability. Species inventory and species richness were comparable with other intertidal areas of the Wadden Sea, but the Jade Bay differs from these areas regarding dominant species. Differences in sediment composition and morphological characteristics (macrotidal versus mesotidal Wadden Sea areas) are discussed for comparison of regional differences.     

NFκB regulates expression of Polo-like kinase 4

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.