Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases,Which Control Stomata Development in Arabidopsis thaliana

Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.     

Brachythecium funkii Schimp. and B. japygum (Głow.) Köckinger & Jan Kučera comb. nov., two Alpine species hitherto included in B. cirrosum (Schwägr.) Schimp.

Morphological and molecular studies support the recognition of three alpine species within the traditional concept of Brachythecium cirrosum, which requires the resurrection of two earlier described taxa, B. funkii and B. japygum, comb. nov., from synonymy. According to nuclear and chloroplast sequence data, the latter two taxa proved to be closely related to B. tommasinii and B. tenuicaule, whereas the affinities of B. cirrosum within Brachythecium are rather unclear. B. funkii is a coarser plant than B. cirrosum and also differs in shorter leaf acumens and much larger lamina cells. On the other hand, B. japygum (better known under the illegitimate name Eurhynchium histrio) can be distinguished from B. cirrosum in the prostrate and densely pinnate to subdendroid growth form, longer leaf acumens and wider lamina cells with lumens wider than walls. All three species also differ from each other in the morphology of the alar cell group. Despite their shared preference for calcareous rocks, the three species show marked differences in habitat requirements.     

One Step Membrane Incorporation of Viral Antigens as a Vaccine Candidate Against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures.

Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines.

The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

High virus-to-cell ratios indicate ongoing production of viruses in deep subsurface sediments

Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (∼14 Ma old) and the most oligotrophic subsurface sediments of the world''s oceans (South Pacific Gyre (SPG)). The numbers of viruses (10⁴–10⁹ cm⁻³, counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.     

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule‐coalescent (GMYC) model‐based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA‐barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large‐scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.     

Spatial and Temporal Dynamics of Exotic Earthworm Communities Along Invasion Fronts in a Temperate Hardwood Forest in South-Central New York (USA)

The invasion of North American forests by exotic earthworms is producing profound ecosystem changes, such as alterations in soil nutrient cycling, and redistribution and loss of soil organic matter. However, the present and future extent of these invasions is difficult to evaluate without a better understanding of the factors that control the distribution and abundance of earthworms in previously non-invaded habitats. In this study, the species composition and short-term dynamics of three exotic earthworm invasion fronts were studied at a northern hardwood forest in south-central New York State (USA). Belt transects were established at each of the three locations to sample from earthworm-invaded areas through transition zones and into invasion front areas. Lumbricus rubellus, L. terrestrisandOctolasion tyrtaeum were the most common species, but their distribution was not homogeneous along the transects. Whereas, L. rubellus was the only species with relatively high adult densities at transition zones and invasion fronts, L. terrestris and O. tyrtaeum occurred mostly in the heavily earthworm-invaded areas and were rare at the invasion fronts. The density of earthworms along the transects decreased by 60–87 from June 2001 to October 2002 and then recovered in 2003 to values similar to those of 2001. This decrease was apparently caused by reduced recruitment of immature earthworms, probably related to the severe drought periods that the study area experienced in 2001 and 2002. Our data suggest that climate and topography, through their effects on soil moisture patterns, can be critical factors controlling the distribution and spread of exotic earthworms in previously non-invaded habitats.     

Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments

Eastern Mediterranean sediments are characterized by the occurrence of distinct, organic-rich layers, called sapropels. These harbour elevated microbial numbers in comparison with adjacent carbon-lean intermediate layers. A recently obtained culture collection from these sediments was composed of 20% of strains closely related to Rhizobium radiobacter, formerly classified as Agrobacterium tumefaciens. To prove and quantify the in situ abundance of R. radiobacter, a highly specific quantitative polymerase chain reaction (PCR) protocol was developed. To convert quantification results into cell numbers, the copy number of rrn operons per genome was determined. Southern hybridization showed that our isolates contained four operons. Finally, quantitative PCR was applied to 45 sediment samples obtained across the eastern Mediterranean. Rhizobium radiobacter was present in 38 of 45 samples indicating an almost ubiquitous distribution. In total, 25-40 000 cells per gram of sediment were detected, corresponding to 0.001-5.1% of the bacterial cells. In general, the relative and absolute abundance of R. radiobacter increased with depth and was higher in sapropels than in intermediate layers. This indicates that R. radiobacter forms an active population in up to 200 000 years old sapropels. The present study shows for the first time that a cultivated subsurface bacterium is highly abundant in this environment.     

Deep biosphere-related bacteria within the subsurface of tidal flat sediments

Biogeochemical and microbiological processes in the upper sediment layers of tidal flats were analysed in many investigations, while deeper zones remained largely unexplored. Therefore, denaturant gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments along the depth profile of up to 5.5 m-long sediment cores was performed in comparison with lithological and geochemical parameters. The investigation revealed that different compartments of the sediment columns were characterized by specific microbial communities. These compartments were analysed by sequencing of 113 DGGE bands. The upper layers down to 160-200 cm were dominated by gamma- and delta-Proteobacteria representing more than 60% of the total number of phylotypes. Underneath, a striking shift in community composition was observed, as the Proteobacteria were replaced by Chloroflexi with more than 60% of all sequences. As sulfate was still available as an electron acceptor in these layers, the abundance of Chloroflexi might be promoted by the electron donor or the quality of the carbon source. The dominance of this group, previously known as green non-sulfur bacteria, indicates the presence of a typical deep-biosphere microbial community in relatively young subsurface sediments. Thus, tidal flats might offer a convenient possibility to study and understand certain aspects of the deep biosphere in general.