We present the experimental demonstration of a subaperture compression scheme achieved in the PETAL (PETawatt Aquitaine Laser) facility. We evidence that by dividing the beam into small subapertures ...fitting the available grating size, the sub-beam can be individually compressed below 1 ps, synchronized below 50 fs and then coherently added thanks to a segmented mirror.
Artificially transforming a scaffold protein into binders often consists of introducing diversity into its natural binding region by directed mutagenesis. We have previously developed the archaeal ...extremophilic Sac7d protein as a scaffold to derive affinity reagents (Affitins) by randomization of only a flat surface, or a flat surface and two short loops with natural lengths. Short loops are believed to contribute to stability of extremophilic proteins, and loop extension has been reported detrimental for the thermal and chemical stabilities of mesophilic proteins. In this work, we wanted to evaluate the possibility of designing target-binding proteins based on Sac7d by using a complementary determining region (CDR). To this aim, we inserted into three different loops a 10 residues CDR from the cAb-Lys3 anti-lysozyme camel antibody. The chimeras obtained were as stable as wild-type (WT) Sac7d at extreme pH and their structural integrity was supported. Chimeras were thermally stable, but with Tms from 60.9 to 66.3°C (cf. 91°C for Sac7d) which shows that loop extension is detrimental for thermal stability of Sac7d. The loop 3 enabled anti-lysozyme activity. These results pave the way for the use of CDR(s) from antibodies and/or extended randomized loop(s) to increase the potential of binding of Affitins.
The Petawatt Aquitaine Laser (PETAL) facility was designed and constructed by the French Commissariat à l'énergie atomique et aux énergies alternatives (CEA) as an additional PW beamline to the Laser ...MegaJoule (LMJ) facility. PETAL energy is limited to 1 kJ at the beginning due to the damage threshold of the final optics. In this paper, we present the commissioning of the PW PETAL beamline. The first kJ shots in the amplifier section with a large spectrum front end, the alignment of the synthetic aperture compression stage and the initial demonstration of the 1.15 PW @ 850 J operations in the compression stage are detailed. Issues encountered relating to damage to optics are also addressed.
The status of the PETAL project is presented in this paper. The global architecture and performances of this facility are detailed with the first experimental results obtained on the LIL facility, ...and with the main steps which will allow shooting in the center of the target chamber. Some technical issues like wavefront correction, damage threshold in femtosecond regime and focusing are presented.
Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ ...receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.
Engineered protein scaffolds have received considerable attention as alternatives to antibodies in both basic and applied research, as they can offer superior biophysical properties often associated ...with a simpler molecular organization. Sac7d has been demonstrated as an effective scaffold for molecular recognition. Here, we used the initial L1 ‘flat surface’ library constructed by randomization of 14 residues, to identify ligands specific for human immunoglobulin G. To challenge the plasticity of the Sac7d protein scaffold, we designed the alternative L2 ‘flat surface & loops’ library whereof only 10 residues are randomized. Representative binders (Affitins) of the two libraries exhibited affinities in the low nanomolar range and were able to recognize different epitopes within human immunoglobulin G. These Affitins were stable up to pH 12 while largely conserving other favorable properties of Sac7d protein, such as high expression yields in Escherichia coli, solubility, thermal stability up to 80.7°C, and acidic stability (pH 0). In agreement with our library designs, mutagenesis study revealed two distinct binding areas, one including loops. Together, our results indicate that the Sac7d scaffold tolerates alternative library designs, which further expands the diversity of Affitins and may provide a general way to create tailored affinity tools for demanding applications.
Directed evolution was used to generate IL-15 mutants with increased solubility and cytoplasmic over-expression in Escherichia coli. A protein solubility selection method was used in which the IL-15 ...gene was expressed as an N-terminal fusion to chloramphenicol acetyltransferase (CAT) as reporter protein. Clones that grew in the presence of high concentrations of chloramphenicol were then screened by ELISA to assay the binding activity of the IL-15-CAT fusion to the IL-15Rα Sushi domain. Two variants of IL-15, M38 and M253, containing five mutations and one mutation respectively, were selected with a dramatic improvement in solubility; the soluble concentration in cell culture was 12- to 18-fold higher, respectively, than for WT IL-15. Characterization of their binding to IL-15Rα and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15Rα and acts as an effective agonist of IL-15.
En este trabajo se presenta una caracterización de la problemática de la educación estadística basada en el concepto del aprendizaje. Se resalta el valor de la motivación vinculada al aprendizaje ...participativo y a la realización de aplicaciones de la metodología estadística a problemas reales, en el contexto de la disciplina del estudiante. Dándose líneas generales para mejorar los cursos de servicio referidos a la elevación de los niveles de aprendizaje.
By cross‐hybridization in low stringency conditions, using a probe derived from an HLA‐DQ beta cDNA clone, we have isolated several chicken genomic DNA clones. These clones were mapped to the major ...histocompatibility complex (MHC) of the chick (B complex) by virtue of their ability to detect restriction enzyme length polymorphisms between congenic lines of chicken. Evidence was obtained for the presence of at least three B‐L beta genes in the chicken genome. The B‐L beta genes are transcribed specifically in tissues containing cells of the B lymphocyte and myeloid lineages and expressing the B‐L antigens. Exons encoding the beta 1, beta 2 and transmembrane domains of a B‐L beta chain have been identified with 63, 66 and 62% similarity with the HLA‐DQ beta sequence. This first isolation of an MHC class II gene outside of the mammalian class provides insight into the evolution of MHC genes based on the comparison of avian and mammalian class II beta chain amino acid and nucleotide sequences.
We have cloned in a cosmid vector four DNA clusters covering 320 kb of the chicken MHC (B complex), including five class II (B‐L) beta genes defining two related isotypic families. Additional B ...complex genes have been revealed using tissue‐specific cDNA probes. A cosmid fragment has been used to isolate a cDNA for a class I (B‐F) transcript. This transcript, that is by far the most divergent known member of the class I gene family, hybridized to six B‐F genes present in the cosmids. One of the clusters was shown to contain two rRNA transcriptional units from the nucleolar organizer region (NOR), marking the telomeric boundary of the B complex. None of the other B complex genes hybridizes to, or has the transcriptional characteristics of mammalian MHC class II alpha or class III genes. The map we have obtained shows that the B complex does not contain well defined class I and class II regions since B‐F and B‐L beta genes are closely associated with unrelated genes. Moreover, class II beta genes are very closely linked to class I genes in two clusters, and to the NOR in a third one.
PDF
