Chiral description of ghost-free massive gravity

We propose and study a new first order version of the ghost-free massive gravity. Instead of metrics or tetrads, it uses a connection together with Plebanski's chiral 2-forms as fundamental variables, rendering the phase space structure similar to that of SU(2) gauge theories. The chiral description simplifies computations of the constraint algebra, and allows us to perform the complete canonical analysis of the system. In particular, we explicitly compute the secondary constraint and carry out the stabilization procedure, thus proving that in general the theory propagates 7 degrees of freedom, consistently with previous claims. Finally, we point out that the description in terms of 2-forms opens the door to an infinite class of ghost-free massive bi-gravity actions. Our results apply directly to Euclidean signature. The reality conditions to be imposed in the Lorentzian signature appear to be more complicated than in the usual gravity case and are left as an open issue.Comment: 26 pages; extended discussion of reality conditions, added reference

Hamiltonian Analysis of non-chiral Plebanski Theory and its Generalizations

We consider non-chiral, full Lorentz group-based Plebanski formulation of general relativity in its version that utilizes the Lagrange multiplier field Phi with "internal" indices. The Hamiltonian analysis of this version of the theory turns out to be simpler than in the previously considered in the literature version with Phi carrying spacetime indices. We then extend the Hamiltonian analysis to a more general class of theories whose action contains scalars invariants constructed from Phi. Such theories have recently been considered in the context of unification of gravity with other forces. We show that these more general theories have six additional propagating degrees of freedom as compared to general relativity, something that has not been appreciated in the literature treating them as being not much different from GR.Comment: 10 page

Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI

Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenylated Rho GTPases. We analyzed the interaction of RhoGDI with the active and inactive forms of prenylated and unprenylated RhoA. We demonstrate that RhoGDI binds the prenylated form of RhoA center dot GDP with unexpectedly high affinity (K-d = 5 pM). The very long half-life of the complex is reduced 25-fold on RhoA activation, with a concomitant reduction in affinity (K-d = 3 nM). The 2.8-angstrom structure of the RhoA center dot guanosine 5'-[beta,gamma-imido] triphosphate (GMPPNP)center dot RhoGDI complex demonstrated that complex formation forces the activated RhoA into a GDP-bound conformation in the absence of nucleotide hydrolysis. We demonstrate that membrane extraction of Rho GTPase by RhoGDI is a thermodynamically favored passive process that operates through a series of progressively tighter intermediates, much like the one that is mediated by RabGDI

Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins

Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic. These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles. Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane. In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins. Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins. However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif. Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways. Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI

Cell-free pipeline for discovery of thermotolerant xylanases and endo-1,4-β-glucanases

The rapid expansion in the number of sequenced genomes and metagenomes provides an exceptional resource for mining of the enzymes with biotechnologically relevant properties. However, the majority of protein production and analysis methods are not sufficiently cost-efficient and scalable to experimentally verify the results of computational genomic mining. Here, we present a pipeline based on Leishmania tarentolae cell-free system that was used to characterize 30 putative thermostable endo-1,4-beta-glucanases and xylanases identified in public genomic databases. In order to analyse the recombinant proteins without purification, novel high-throughput assays for glucanase and xylanase activities were developed. The assays rely on solubilisation of labelled particulate substrates performed in multiwell plates. Using this approach both acidophilic and thermophilic enzymes were identified. The developed approach enables rapid discovery of new biotechnologically useful enzymes

A New Spin Foam Model for 4d Gravity

Starting from Plebanski formulation of gravity as a constrained BF theory we propose a new spin foam model for 4d Riemannian quantum gravity that generalises the well-known Barrett-Crane model and resolves the inherent to it ultra-locality problem. The BF formulation of 4d gravity possesses two sectors: gravitational and topological ones. The model presented here is shown to give a quantization of the gravitational sector, and is dual to the recently proposed spin foam model of Engle et al. which, we show, corresponds to the topological sector. Our methods allow us to introduce the Immirzi parameter into the framework of spin foam quantisation. We generalize some of our considerations to the Lorentzian setting and obtain a new spin foam model in that context as well.Comment: 40 pages; (v2) published versio

Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research

Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed

Novel antiinflammatory biologics shaped by parasite–host coevolution

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host’s immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm’s excretory–secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases