Light-Emitting Channelrhodopsins for Combined Optogenetic and Chemical-Genetic Control of Neurons

Manipulation of neuronal activity through genetically targeted actuator molecules is a powerful approach for studying information flow in the brain. In these approaches the genetically targeted component, a receptor or a channel, is activated either by a small molecule (chemical genetics) or by light from a physical source (optogenetics). We developed a hybrid technology that allows control of the same neurons by both optogenetic and chemical genetic means. The approach is based on engineered chimeric fusions of a light-generating protein (luciferase) to a light-activated ion channel (channelrhodopsin). Ionic currents then can be activated by bioluminescence upon activation of luciferase by its substrate, coelenterazine (CTZ), as well as by external light. In cell lines, expression of the fusion of Gaussia luciferase to Channelrhodopsin-2 yielded photocurrents in response to CTZ. Larger photocurrents were produced by fusing the luciferase to Volvox Channelrhodopsin-1. This version allowed chemical modulation of neuronal activity when expressed in cultured neurons: CTZ treatment shifted neuronal responses to injected currents and sensitized neurons to fire action potentials in response to subthreshold synaptic inputs. These luminescent channelrhodopsins - or luminopsins – preserve the advantages of light-activated ion channels, while extending their capabilities. Our proof-of-principle results suggest that this novel class of tools can be improved and extended in numerous ways.     

Import and export of biological samples from tropical countries–considerations and guidelines for research teams

‘Biodiversity’ is increasingly perceived as an important resource for research and conservation, but also for economy. Conservation, access and sustainable use of biodiversity (genetic resources, species, samples) are negotiated on different political levels, resulting in an internationally binding legal framework. Resulting legislation is binding for all parties involved in biological sampling, i.e. researches and (and in italics) countries, and especially applies for tissue or DNA samples and dervied products thereof. Understanding and awareness of export and import permits for biological samples is increasingly important for biologists to perform research projects legally and timely. Nevertheless, some biologists are still exporting and importing biological samples ignoring or non-compliant with national and international legislation, conventions, and regulations. Resulting difficulties may not only cause serious problems during field work, but may also delay the export, import or exchange of samples. Comprehensive a priori information regarding legal requirements helps to avoid or at least diminish potential problems. We identified four major factors facilitating export/import permits: (1) good personal (mutually trusted) contacts in the country of origin, (2) understanding and compliance with all relevant laws and regulations; (3) access to information regarding knowledge on permits, regulations and laws including their circulation within the researcher communities; and (4) access to consistent and up to date regulations     

Die Weichbodengemeinschaft vor der Elbemündung unter dem Einfluß der Klärschlammverklappung. II. Die Gemeinschaftsstruktur im saisonalen Wechsel

Macrobenthic fauna and sediment parameters were studied at 40 stations representing a 494 km² survey area in the eastern part of the German Bight and encompassing the sewage sludge dumping site. Data were taken in July 1980, one month after dumping ended, and in April 1981. pH-value, redox potential of the interstitial water, amount of sand in the sediment as well as total number of species and diversity were lower in the dumping area than outside, whereas alkalinity and water content of the sediment were higher. Due to mass development of a few species around the dumping site, biomass and number of individuals were high in July. Abundance and biomass values were lower in April. 2 communities are described and related to abiotic parameters such as sediment structure. Due to disturbance effects of sewage sludge, 2 stages of the second successional phase (Pearson & Rosenberg, 1978) were ascertained, described by one of these communities each time. The first faunal community, characterized by anAbra alba assemblage, describes an early stage of the second successional phase. In summer (July) theAbra alba community showed a wider spatial range than in spring. The other, dominated byNucula nitida, indicated a later stage of the second succession phase that was passing to the third phase.      

Recombinant expression of tatABC and tatAC results in the formation of interacting cytoplasmic TatA tubes in Escherichia coli

The twin-arginine translocation (Tat) system of bacteria and plant plastids serves to translocate folded proteins across energized biological membranes. In Escherichia coli, the three components TatA, TatB, and TatC mediate this membrane passage. Here we demonstrate that TatA can assemble to form clusters of tube-like structures in vivo. While the presence of TatC is essential for their formation, TatB is not required. The TatA tubes have uniform outer and inner diameters of about 11.5 nm and 6.7 nm, respectively. They align to form a crystalline-like structure in which each tube is surrounded by six TatA tubes. The tube structures become easily detectable even at only a 15-fold overexpression of the tatABC genes. The TatA tubes could also be visualized by fluorescence when untagged TatA was mixed with low amounts of TatA-GFP. The structures were often found in contact with the cell poles. Because TatC is most likely polar in E. coli, as demonstrated by a RR-dependent targeting of translocation-incompatible Tat substrates to the cell poles, and because TatC is required for the formation of aligned TatA tubes, it is proposed that the TatA tubes are initiated at polarly localized TatC.     

Plasmodium yoelii sporozoites modulate cytokine profile and induce apoptosis in murine Kupffer cells

Plasmodium sporozoites traverse Kupffer cells on their way into the liver. Sporozoite contact does not elicit a respiratory burst in these hepatic macrophages and blocks the formation of reactive oxygen species in response to secondary stimuli via elevation of the intracellular cAMP concentration. Here we show that increasing the cAMP level with dibutyryl cyclic adenosine monophosphate (db-cAMP) or isobutylmethylxanthine (IBMX) also modulates cytokine secretion in murine Kupffer cells towards an overall anti-inflammatory profile. Stimulation of Plasmodium yoelii sporozoite-exposed Kupffer cells with lipopolysaccharide or IFN-γ reveals down-modulation of TNF-α, IL-6 and MCP-1, and up-regulation of IL-10. Prerequisite for this shift of the cytokine profile are parasite viability and contact with Kupffer cells, but not invasion. Whilst sporozoite-exposed Kupffer cells become TUNEL-positive and exhibit other signs of apoptotic death such as membrane blebbing, nuclear condensation and fragmentation, sporozoites remain intact and appear to transform to early exo-erythrocytic forms in Kupffer cell cultures. Together, the in vitro data indicate that Plasmodium possesses mechanisms to render Kupffer cells insensitive to pro-inflammatory stimuli and eventually eliminates these macrophages by forcing them into programmed cell death.     

Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR

The ATP binding cassette (ABC) transporter LmrA from Lactococcus lactis transports cytotoxic molecules at the expense of ATP. Molecular and kinetic details of LmrA can be assessed by solid-state nuclear magnetic resonance (ssNMR), if functional reconstitution at a high protein-lipid ratio can be achieved and the kinetic rate constants are small enough. In order to follow ATP hydrolysis directly by 31P-magic angle spinning (MAS) nuclear magnetic resonance (NMR), we generated such conditions by reconstituting LmrA-dK388, a mutant with slower ATP turnover rate, at a protein-lipid ration of 1:150. By analysing time-resolved 31P spectra, protein activity has been directly assessed. These data demonstrate the general possibility to perform ssNMR studies on a fully active full length ABC transporter and also form the foundation for further kinetic studies on LmrA by NMR.     

In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.     

Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein, as well as with an electron-transfer function via thiol-disulfide interchanges.     

Mucosal Immunization of Cynomolgus Macaques with the VSVΔG/ZEBOVGP Vaccine Stimulates Strong Ebola GP-Specific Immune Responses

Background

Zaire ebolavirus (ZEBOV) produces a lethal viral hemorrhagic fever in humans and non-human primates.

Methodology/Principal Findings

We demonstrate that the VSVΔG/ZEBOVGP vaccine given 28 days pre-challenge either intranasally (IN), orally (OR), or intramuscularly (IM) protects non-human primates against a lethal systemic challenge of ZEBOV, and induces cellular and humoral immune responses. We demonstrated that ZEBOVGP-specific T-cell and humoral responses induced in the IN and OR groups, following an immunization and challenge, produced the most IFN-γ and IL-2 secreting cells, and long term memory responses.

Conclusions/Significance

We have shown conclusively that mucosal immunization can protect from systemic ZEBOV challenge and that mucosal delivery, particularly IN immunization, seems to be more potent than IM injection in the immune parameters we have tested. Mucosal immunization would be a huge benefit in any emergency mass vaccination campaign during a natural outbreak, or following intentional release, or for mucosal immunization of great apes in the wild.