Production of a Functional Catalytic Antibody ScFv-NusA Fusion Protein in Bacterial Cytoplasm

Functional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selectio

An efficient one-step site-directed and site-saturation mutagenesis protocol

We have developed a new primer design method based on the QuickChange™ site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange™ protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation librarie

Monitoring resilience in a rook-managed containerized cloud storage system

Distributed cloud storage solutions are currently gaining high momentum in industry and academia. The enterprise data volume growth and the recent tendency to move as much as possible data to the cloud is strongly stimulating the storage market growth. In this context, and as a main requirement for cloud native applications, it is of utmost importance to guarantee resilience of the deployed applications and the infrastructure. Indeed, with failures frequently occurring, a storage system should quickly recover to guarantee service availability. In this paper, we focus on containerized cloud storage, proposing a resilience monitoring solution for the recently developed Rook storage operator. While, Rook brings storage systems into a cloud-native container platform, in this paper we design an additional module to monitor and evaluate the resilience of the Rook-based system. Our proposed module is validated in a production environment, with software components generating a constant load and a controlled removal of system elements to evaluate the self-healing capability of the storage system. Failure recovery time revealed to be 41 and 142 seconds on average for a 32GB and a 215GB object storage device respectively

The ethical and legal aspects of palliative sedation in severely brain-injured patients: a French perspective

To fulfill their crucial duty of relieving suffering in their patients, physicians may have to administer palliative sedation when they implement treatment-limitation decisions such as the withdrawal of life-supporting interventions in patients with poor prognosis chronic severe brain injury. The issue of palliative sedation deserves particular attention in adults with serious brain injuries and in neonates with severe and irreversible brain lesions, who are unable to express pain or to state their wishes. In France, treatment limitation decisions for these patients are left to the physicians. Treatment-limitation decisions are made collegially, based on the presence of irreversible brain lesions responsible for chronic severe disorders of consciousness. Before these decisions are implemented, they are communicated to the relatives. Because the presence and severity of pain cannot be assessed in these patients, palliative analgesia and/or sedation should be administered. However, palliative sedation is a complex strategy that requires safeguards to prevent a drift toward hastening death or performing covert euthanasia. In addition to the law on patients' rights at the end of life passed in France on April 22, 2005, a recent revision of Article 37 of the French code of medical ethics both acknowledges that treatment-limitation decisions and palliative sedation may be required in patients with severe brain injuries and provides legal and ethical safeguards against a shift towards euthanasia. This legislation may hold value as a model for other countries where euthanasia is illegal and for countries such as Belgium and Netherlands where euthanasia is legal but not allowed in patients incapable of asking for euthanasia but in whom a treatment limitation decision has been made

A series of Notch3 mutations in CADASIL; insights from 3D molecular modelling and evolutionary analyses

CADASIL disease belongs to the group of rare diseases. It is well established that the Notch3 protein is primarily responsible for the development of CADASIL syndrome. Herein, we attempt to shed light to the actual molecular mechanism underlying CADASIL via insights that we have from preliminary in silico and proteomics studies on the Notch3 protein. At the moment, we are aware of a series of Notch3 point mutations that promote CADASIL. In this direction, we investigate the nature, extent, physicochemical and structural significance of the mutant species in an effort to identify the underlying mechanism of Notch3 role and implications in cell signal transduction. Overall, our in silico study has revealed a rather complex molecular mechanism of Notch3 on the structural level; depending of the nature and position of each mutation, a consensus significant loss of beta-sheet structure is observed throughout all in silico modeled mutant/wild type biological systems

Activity of ulilysin, an archaeal PAPP-A-related gelatinase and IGFBP protease

Human growth and development are conditioned by insulin-like growth factors (IGFs), which have also implications in pathology. Most IGF molecules are sequestered by IGF-binding proteins (IGFBPs) so that exertion of IGF activity requires disturbance of these complexes. This is achieved by proteolysis mediated by IGFBP proteases, among which the best characterised is human PAPP-A, the first member of the pappalysin family of metzincins. We have previously identified and studied the only archaeal homologue found to date, Methanosarcina acetivorans ulilysin. This is a proteolytically functional enzyme encompassing a pappalysin catalytic domain and a pro-domain involved in maintenance of latency of the zymogen, proulilysin. Once activated, the protein hydrolyses IGFBP-2 to -6 and insulin chain β in vitro. We report here that ulilysin is also active against several other substrates, viz (azo)casein, azoalbumin, and extracellular matrix components. Ulilysin has gelatinolytic but not collagenolytic activity. Moreover, the proteolysis-resistant skeletal proteins actin and elastin are also cleaved, as is fibrinogen, but not plasmin and α1-antitrypsin from the blood coagulation cascade. Ulilysin develops optimal activity at pH 7.5 and strictly requires peptide bonds preceding an arginine residue, as determined by means of a novel fluorescence resonance energy transfer assay, thus pointing to biotechnological applications as an enzyme complementary to trypsi

Update of D3/D7-Brane Inflation on K3 x T^2/Z_2

We update the D3/D7-brane inflation model on K3 x T^2/Z_2 with branes and fluxes. For this purpose, we study the low energy theory including g_s corrections to the gaugino condensate superpotential that stabilizes the K3 volume modulus. The gauge kinetic function is verified to become holomorphic when the original N=2 supersymmetry is spontaneously broken to N=1 by bulk fluxes. From the underlying classical N=2 supergravity, the theory inherits a shift symmetry which provides the inflaton with a naturally flat potential. We analyze the fate of this shift symmetry after the inclusion of quantum corrections. The field range of the inflaton is found to depend significantly on the complex structure of the torus but is independent of its volume. This allows for a large kinematical field range for the inflaton. Furthermore, we show that the D3/D7 model may lead to a realization of the recent CMB fit by Hindmarsh et al. with an 11% contribution from cosmic strings and a spectral index close to n_s=1. On the other hand, by a slight change of the parameters of the model one can strongly suppress the cosmic string contribution and reduce the spectral index n_s to fit the WMAP5 data in the absence of cosmic strings. We also demonstrate that the inclusion of quantum corrections allows for a regime of eternal D3/D7 inflation.Comment: LaTeX2e, 55 pages + appendices, 8 figures; v3: added appendix F and a note at the end of the conclusions in order to clarify the relation of our results to the recent work by Burgess et al. (arXiv:0811.1503

Core Values that Influence the Patient—Healthcare Professional Power Dynamic: Steering Interaction towards Partnership

Healthcare has long been marked by the authoritative-physician–passive-patient interaction, with patients seeking help and physicians seeking to restore patients back to health. However, lobalisation, social movements, and technological advancements are transforming the nature of this relationship. We aim to identify core values that influence the power dynamic betweenpatients and healthcare professionals, and determine how to steer these interactions towards partnership, a more suitable approach to current healthcare needs. Patients with physical chronic diseases (10 men, 18 women) and healthcare professionals (11 men, 12 women) were interviewed, sessions transcribed, and the framework method used to thematically analyse the data. Validation was done through analyst triangulation and member check recheck. Core values identified as influencing the patient-healthcare professional power dynamic include: (A) values that empower patients (acceptance of diagnosis and autonomy); (B) values unique to healthcare professionals (HCPs) (acknowledging patients experiential knowledge and including patients in the therapeutic process); and (C) shared capitals related to their interactions (communication, information sharing and exchange, collaboration, and mutual commitment). These interdependent core values can be considered prerequisites to the implementation of the patient-as-partner approach in healthcare. Partnership would imply a paradigm shift such that stakeholders systematically examine each other’s perspective, motivations, capabilities, and goals, and then adapt their interactions in this accord, for optimal outcome

Cosmology of the Tachyon in Brane Inflation

In certain implementations of the brane inflationary paradigm, the exit from inflation occurs when the branes annihilate through tachyon condensation. We investigate various cosmological effects produced by this tachyonic era. We find that only a very small region of the parameter space (corresponding to slow-roll with tiny inflaton mass) allows for the tachyon to contribute some e-folds to inflation. In addition, non-adiabatic density perturbations are generated at the end of inflation. When the brane is moving relativistically this contribution can be of the same order as fluctuations produced 55 e-folds before the end of inflation. The additional contribution is very nearly scale-invariant and enhances the tensor/scalar ratio. Additional non-gaussianities will also be generated, sharpening current constraints on DBI-type models which already predict a significantly non-gaussian signal.Comment: 30 pages, 2 figures; v3, minor revision, JCAP versio