Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.     

Chloroperoxidase-catalyzed cyclodimerization of methyl (2E)-2,4-pentadienoate: a [4+2] cycloaddition product

The chloroperoxidase (CPO)-catalyzed oxidation of the methyl (2E)-2,4-pentadienoate gives the terminal double bond epoxide (25%) and a cyclodimerization compound (63%) as the major products.     

Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice.

Fibroblast growth factor-23 (FGF-23), a recently identified molecule that is mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR), appears to be involved in the regulation of phosphate homeostasis. Although increased levels of circulating FGF-23 were detected in patients with different phosphate-wasting disorders such as oncogenic osteomalacia (OOM) and X-linked hypophosphatemia (XLH), it is not yet clear whether FGF-23 is directly responsible for the abnormal regulation of mineral ion homeostasis and consequently bone development. To address some of these unresolved questions, we generated a mouse model, in which the entire Fgf-23 gene was replaced with the lacZ gene. Fgf-23 null (Fgf-23-/-) mice showed signs of growth retardation by day 17, developed severe hyperphosphatemia with elevated serum 1,25(OH)2D3 levels, and died by 13 weeks of age. Hyperphosphatemia in Fgf-23-/- mice was accompanied by skeletal abnormalities, as demonstrated by histological, molecular, and various other morphometric analyses. Fgf-23-/-) mice had increased total-body bone mineral content (BMC) but decreased bone mineral density (BMD) of the limbs. Overall, Fgf-23-/- mice exhibited increased mineralization, but also accumulation of unmineralized osteoid leading to marked limb deformities. Moreover, Fgf-23-/- mice showed excessive mineralization in soft tissues, including heart and kidney. To further expand our understanding regarding the role of Fgf-23 in phosphate homeostasis and skeletal mineralization, we crossed Fgf-23-/- animals with Hyp mice, the murine equivalent of XLH. Interestingly, Hyp males lacking both Fgf-23 alleles were indistinguishable from Fgf-23/-/ mice, both in terms of serum phosphate levels and skeletal changes, suggesting that Fgf-23 is upstream of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and that the increased plasma Fgf-23 levels in Hyp mice (and in XLH patients) may be at least partially responsible for the phosphate imbalance in this disorder.     

POLLINATION ECOLOGY OF LABIATAE IN A PHRYGANIC (EAST MEDITERRANEAN) ECOSYSTEM

This study was conducted in a phryganic (East Mediterranean) ecosystem at Daphni, near Athens, Greece. The Labiatae, represented by ten species belonging to nine genera, dominate in this ecosystem type. They flower from February to July. Both flowering time and nectar quantity are related to the species ability to tolerate intense water stress. Labiatae are visited by 201 insect species. Of these, 43 are exclusively supported by the family and 37 are monotropous. Solitary bees (mainly Anthophoridae, Megachilidae, Halictidae) constitute 47.3% of pollinators. The family is important in hosting specialized bees (15 species) in phrygana, particularly late in the flowering season. Labiatae species form two equally represented groups in this system; namely, the late winter-early spring (early) flowering, visited by relatively few pollinator species, and the late spring-summer (late) flowering species, visited by numerous pollinators. This temporal distinction is accompanied by different pollination profiles that include duration of anthesis, reward to pollinators, floral attractiveness, and flower character differentiation. All of these attributes are maximized in the early flowering period. This strategy suggests a mechanism for resource partitioning at a time when the pollinator resource is limited and competition for the services of pollinators is expected to be intense. Contrary to the current theory concerning cornucopian species, the copiously rewarding flowers of Labiatae in phrygana are not those abundantly serviced by pollinators.     

Iterative subtractive binning of freshwater chronoseries metagenomes identifies over 400 novel species and their ecologic preferences

Recent advances in sequencing technology and bioinformatic pipelines have allowed unprecedented access to the genomes of yet-uncultivated microorganisms from diverse environments. However, the catalogue of freshwater genomes remains limited, and most genome recovery attempts in freshwater ecosystems have only targeted specific taxa. Here, we present a genome recovery pipeline incorporating iterative subtractive binning, and apply it to a time series of 100 metagenomic datasets from seven connected lakes and estuaries along the Chattahoochee River (Southeastern USA). Our set of metagenome-assembled genomes (MAGs) represents >400 yet-unnamed genomospecies, substantially increasing the number of high-quality MAGs from freshwater lakes. We propose names for two novel species: ‘Candidatus Elulimicrobium humile’ (‘Ca. Elulimicrobiota’, ‘Patescibacteria’) and ‘Candidatus Aquidulcis frankliniae’ (‘Chloroflexi’). Collectively, our MAGs represented about half of the total microbial community at any sampling point. To evaluate the prevalence of these genomospecies in the chronoseries, we introduce methodologies to estimate relative abundance and habitat preference that control for uneven genome quality and sample representation. We demonstrate high degrees of habitat-specialization and endemicity for most genomospecies in the Chattahoochee lakes. Wider ecological ranges characterized smaller genomes with higher coding densities, indicating an overall advantage of smaller, more compact genomes for cosmopolitan distributions.     

Neisseria meningitidis-induced death of cerebrovascular endothelium: mechanisms triggering transcriptional activation of inducible nitric oxide synthase

The intense host response to meningococcus reflects marked functional and morphological alterations in blood-brain barriers. We showed previously that mouse-derived cerebrovascular endothelium responded to meningococcal lysates with a robust nitric oxide (NO) response, resulting in the loss of cell viability. To understand how the NO synthase-2 gene in endothelium is activated by meningococcus, we investigated upstream roles for specific protein kinases. Using known kinase inhibitors, and measuring both mRNA expression and nitrite release, we found MAPK/ERK kinase (MEK)2, p38 kinase and phosphoinositide 3-kinase (but not MEK1 or phospholipase C) to be implicated in the NO synthase-2 response. Recruitment of these kinases by meningococcus did not depend on the prior release of the proinflammatory cytokines tumour necrosis factor alpha or interleukin-1beta from endothelium. These endothelial cells were found to express toll-like receptors (TLR) 2, 4 and 9 and antibodies directed against TLR 2 and 4 (but not TLR 9) blocked the NO synthase-2 response to meningococcus. Both meningococcus-induced translocation of nuclear factor-kB (NF-kB) and endothelial cell death were blocked by a known inhibitor of p38 kinase. Calpain inhibitor-1 blocked the NO synthase-2 response to meningococcus, which is further evidence of a role for NF-kB.     

Heat-induced degradation of overexpressed glucocorticoid receptor Separate protective roles of hsp90 and hsp70

The glucocorticoid receptor (GR) occurs in cells in the form of a hormone-responsive complex (HRC) with hsp90. The HRC is dynamic, with hsp90 constantly directing disassembly, and hsp70, assisted by hsp90, driving reassembly. WCL2 cells stably overexpress GR to an extent that reduces the excess of hsp90 and hsp70 over GR by about 10-fold, compared to the ratio in HeLa cells. Yet the half-lives of the HRC in WCL2 and HeLa cells are comparable. As a result, the rate of assembly in WCL2 is overwhelmed by accumulation of the non-hormone-binding form of GR in its complex with hsp70 and hsp90. This form comprised some 50% of total GR in WCL2 cells. When the cells were heated to 44 degrees C, the hormone-binding activity and solubility of GR fell in parallel, and the receptor formed heavy aggregates by sequestering large amounts of hsp70. About 40% of this aggregated receptor was degraded in cells recovering at 37 degrees C in the presence of cycloheximide. Concentration of GR protein increased with increasing induction of hsp70 following exposure to 41-44 degrees C. However, balance between hormone-binding and inert forms of GR could shift in either direction in response to the increase or decrease of hsp90 induction, depending on the temperature. Suppression of degradation following re-exposure of the cells to 44 degrees C correlated better with induction of hsp90 than hsp70. We infer that sequestration of hsp70 by heat-unfolded receptor is the primary factor opposing degradation, while induction of hsp90 acts to further suppress degradation by accelerating HRC assembly.     

Comparison of closely related,uncultivated Coxiella tick endosymbiont population genomes reveals clues about the mechanisms of symbiosis

Understanding the symbiotic interaction between Coxiella‐like endosymbionts (CLE) and their tick hosts is challenging due to lack of isolates and difficulties in tick functional assays. Here we sequenced the metagenome of a CLE population from wild Rhipicephalus sanguineus ticks (CRs) and compared it to the previously published genome of its close relative, CLE of R. turanicus (CRt). The tick hosts are closely related sympatric species, and their two endosymbiont genomes are highly similar with only minor differences in gene content. Both genomes encode numerous pseudogenes, consistent with an ongoing genome reduction process. In silico flux balance metabolic analysis (FBA) revealed the excess production of L‐proline for both genomes, indicating a possible proline transport from Coxiella to the tick. Additionally, both CR genomes encode multiple copies of the proline/betaine transporter, proP gene. Modelling additional Coxiellaceae members including other tick CLE, did not identify proline as an excreted metabolite. Although both CRs and CRt genomes encode intact B vitamin synthesis pathway genes, which are presumed to underlay the mechanism of CLE‐tick symbiosis, the FBA analysis indicated no changes for their products. Therefore, this study provides new testable hypotheses for the symbiosis mechanism and a better understanding of CLE genome evolution and diversity.     

Corneal-Committed Cells Restore the Stem Cell Pool and Tissue Boundary following Injury

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