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.     

Sustainable control of mosquito larvae in the field by the combined actions of the biological insecticide Bti and natural competitors

Integrated management of mosquitoes is becoming increasingly important, particularly in relation to avoiding recolonization of ponds after larvicide treatment. We conducted for the first time field experiments that involved exposing natural populations of the mosquito species Culex pipiens to: a) application of the biological insecticide Bacillus thuringiensis israelensis (Bti), b) the introduction of natural competitors (a crustacean community composed mainly of Daphnia spp.), or c) a combined treatment that involved both introduction of a crustacean community and the application of Bti. The treatment that involved only the introduction of crustaceans had no significant effect on mosquito larval populations, while treatment with Bti alone caused only a significant reduction in the abundance of mosquito larvae in the short‐term (within 3–10 days after treatment). In contrast, the combined treatment rapidly reduced the abundance of mosquito larvae, which remained low throughout the entire observation period of 28 days. Growth of the introduced crustacean communities was favored by the immediate reduction in the abundance of mosquito larvae following Bti administration, thus preventing recolonization of ponds by mosquito larvae at the late period (days 14–28 after treatment). Both competition and the temporal order of establishment of different species are hence important mechanisms for efficient and sustainable mosquito control.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

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.     

Nematode succession at deep-sea hydrothermal vents after a recent volcanic eruption with the description of two dominant species

Nematodes are very common in the deep sea and are an important component of deep-sea hydrothermal vent communities. In early 2006, the eruption of the underwater volcano at 9°50’N East Pacific Rise wiped out almost the entire faunal communities of the area. This provided us with the opportunity to study nematode primary succession at vents as well as on adjacent seafloor basalt. Nematode abundance and richness were extremely low at all studied sites in late 2006 and 2007, and increased only slightly in 2009. Interestingly, the most abundant species during early succession were also prominent in this area prior to the eruption. Our results show that nematodes are extremely influenced by volcanic eruptions and need a long period of time to colonize the lava-flooded area in greater numbers and richness. We hypothesize that low food availability on the young bare basalt and harsh environmental conditions at early succession vent sites might hinder a more successful nematode establishment. In addition to the newly established active vent sites we also studied an inactive vent site that was not directly hit by the eruption but whose vent fluid had ceased after the eruption. At this inactive and older vent, diversity was also relatively low but was higher than at the younger, newly established sites. In addition to the ecological analyses, we here describe the two most abundant species found at inactive vents, namely Neochromadora aff. poecilosoma De Mann 1893 and Linhomoeus caudipapillosus sp. n.     

The Effect of Music on the Human Stress Response

Background

Music listening has been suggested to beneficially impact health via stress-reducing effects. However, the existing literature presents itself with a limited number of investigations and with discrepancies in reported findings that may result from methodological shortcomings (e.g. small sample size, no valid stressor). It was the aim of the current study to address this gap in knowledge and overcome previous shortcomings by thoroughly examining music effects across endocrine, autonomic, cognitive, and emotional domains of the human stress response.

Methods

Sixty healthy female volunteers (mean age = 25 years) were exposed to a standardized psychosocial stress test after having been randomly assigned to one of three different conditions prior to the stress test: 1) relaxing music (‘Miserere’, Allegri) (RM), 2) sound of rippling water (SW), and 3) rest without acoustic stimulation (R). Salivary cortisol and salivary alpha-amylase (sAA), heart rate (HR), respiratory sinus arrhythmia (RSA), subjective stress perception and anxiety were repeatedly assessed in all subjects. We hypothesized that listening to RM prior to the stress test, compared to SW or R would result in a decreased stress response across all measured parameters.

Results

The three conditions significantly differed regarding cortisol response (p = 0.025) to the stressor, with highest concentrations in the RM and lowest in the SW condition. After the stressor, sAA (p=0.026) baseline values were reached considerably faster in the RM group than in the R group. HR and psychological measures did not significantly differ between groups.

Conclusion

Our findings indicate that music listening impacted the psychobiological stress system. Listening to music prior to a standardized stressor predominantly affected the autonomic nervous system (in terms of a faster recovery), and to a lesser degree the endocrine and psychological stress response. These findings may help better understanding the beneficial effects of music on the human body.     

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.     

Methane-Carbon Flow into the Benthic Food Web at Cold Seeps – A Case Study from the Costa Rica Subduction Zone

Cold seep ecosystems can support enormous biomasses of free-living and symbiotic chemoautotrophic organisms that get their energy from the oxidation of methane or sulfide. Most of this biomass derives from animals that are associated with bacterial symbionts, which are able to metabolize the chemical resources provided by the seeping fluids. Often these systems also harbor dense accumulations of non-symbiotic megafauna, which can be relevant in exporting chemosynthetically fixed carbon from seeps to the surrounding deep sea. Here we investigated the carbon sources of lithodid crabs (Paralomis sp.) feeding on thiotrophic bacterial mats at an active mud volcano at the Costa Rica subduction zone. To evaluate the dietary carbon source of the crabs, we compared the microbial community in stomach contents with surface sediments covered by microbial mats. The stomach content analyses revealed a dominance of epsilonproteobacterial 16S rRNA gene sequences related to the free-living and epibiotic sulfur oxidiser Sulfurovum sp. We also found Sulfurovum sp. as well as members of the genera Arcobacter and Sulfurimonas in mat-covered surface sediments where Epsilonproteobacteria were highly abundant constituting 10% of total cells. Furthermore, we detected substantial amounts of bacterial fatty acids such as i-C15∶0 and C17∶1ω6c with stable carbon isotope compositions as low as −53‰ in the stomach and muscle tissue. These results indicate that the white microbial mats at Mound 12 are comprised of Epsilonproteobacteria and that microbial mat-derived carbon provides an important contribution to the crab''s nutrition. In addition, our lipid analyses also suggest that the crabs feed on other ¹³C-depleted organic matter sources, possibly symbiotic megafauna as well as on photosynthetic carbon sources such as sedimentary detritus.     

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.