Surface display of a single-domain antibody library on Gram-positive bacteria

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10⁷ camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.     

Genetically modified Plasmodium parasites as a protective experimental malaria vaccine

Malaria is a mosquito-borne disease that is transmitted by inoculation of the Plasmodium parasite sporozoite stage. Sporozoites invade hepatocytes, transform into liver stages, and subsequent liver-stage development ultimately results in release of pathogenic merozoites. Liver stages of the parasite are a prime target for malaria vaccines because they can be completely eliminated by sterilizing immune responses, thereby preventing malarial infection. Using expression profiling, we previously identified genes that are only expressed in the pre-erythrocytic stages of the parasite. Here, we show by reverse genetics that one identified gene, UIS3 (upregulated in infective sporozoites gene 3), is essential for early liver-stage development. uis3-deficient sporozoites infect hepatocytes but are unable to establish blood-stage infections in vivo, and thus do not lead to disease. Immunization with uis3-deficient sporozoites confers complete protection against infectious sporozoite challenge in a rodent malaria model. This protection is sustained and stage specific. Our findings demonstrate that a safe and effective, genetically attenuated whole-organism malaria vaccine is possible.     

Emergence: A Systems Theory’s Challenge to Ethics

The paper reconstructs Niklas Luhmann’s diagnosis of the dysfunctional character of moral communication in the modern society by emphasizing the emergent character of today’s moral problems. In the systems-theoretic literature, emergence means the irreducibility of the properties of the whole to the characteristics of its parts. Two arguments have been advanced. First, the dysfunctional character of moral communication has been traced back to the emergent character of many moral problems. Moral communication has thus been shown to be not inherently dysfunctional, but rather needful of semantic forms that take account of the emergent properties of the economic and other social systems. Second, these properties highlight the moral aspect of the precariousness of system–environment relations as seen by Luhmann. As a moral problem, this precariousness can be resolved through greater sensitivity of social systems to their environment, social and natural alike. Accordingly, the emergent properties of the economic and other social systems can be captured by recasting the concept of responsibility as the individual-level or organizational-level projection of the environmental sensitivity of these systems.     

Progressive field-state collapse and quantum non-demolition photon counting

The irreversible evolution of a microscopic system under measurement is a central feature of quantum theory. From an initial state generally exhibiting quantum uncertainty in the measured observable, the system is projected into a state in which this observable becomes precisely known. Its value is random, with a probability determined by the initial system's state. The evolution induced by measurement (known as 'state collapse') can be progressive, accumulating the effects of elementary state changes. Here we report the observation of such a step-by-step collapse by non-destructively measuring the photon number of a field stored in a cavity. Atoms behaving as microscopic clocks cross the cavity successively. By measuring the light-induced alterations of the clock rate, information is progressively extracted, until the initially uncertain photon number converges to an integer. The suppression of the photon number spread is demonstrated by correlations between repeated measurements. The procedure illustrates all the postulates of quantum measurement (state collapse, statistical results and repeatability) and should facilitate studies of non-classical fields trapped in cavities.     

Structure of the apoptotic protease-activating factor 1 bound to ADP

Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.     

Orai1 is an essential pore subunit of the CRAC channel

Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.     

Ocean circulation and climate during the past 120,000 years

Oceans cover more than two-thirds of our blue planet. The waters move in a global circulation system, driven by subtle density differences and transporting huge amounts of heat. Ocean circulation is thus an active and highly nonlinear player in the global climate game. Increasingly clear evidence implicates ocean circulation in abrupt and dramatic climate shifts, such as sudden temperature changes in Greenland on the order of 5-10 degrees C and massive surges of icebergs into the North Atlantic Ocean --events that have occurred repeatedly during the last glacial cycle.