The effects of N-terminal insertion into VSV-G of an scFv peptide

Recombinant retroviruses, including lentiviruses, are the most widely used vectors for both in vitro and in vivo stable gene transfer. However, the inability to selectively deliver transgenes into cells of interest limits the use of this technology. Due to its wide tropism, stability and ability to pseudotype a range of viral vectors, vesicular stomatitis virus G protein (VSV-G) is the most commonly used pseudotyping protein. Here, we attempted to engineer this protein for targeting purposes. Chimaeric VSV-G proteins were constructed by linking a cell-directing single-chain antibody (scFv) to its N-terminal. We show that the chimaeric VSV-G molecules can integrate into retroviral and lentiviral particles. HIV-1 particles pseudotyped with VSV-G linked to an scFv against human Major Histocompatibility Complex class I (MHC-I) bind strongly and specifically to human cells. Also, this novel molecule preferentially drives lentiviral transduction of human cells, although the titre is considerably lower that viruses pseudotyped with VSV-G. This is likely due to the inefficient fusion activity of the modified protein. To our knowledge, this is the first report where VSV-G was successfully engineered to include a large (253 amino acids) exogenous peptide and where attempts were made to change the infection profile of VSV-G pseudotyped vectors

Chorioallantoic membrane assay as model for angiogenesis in tissue engineering : focus on stem cells

Tissue engineering aims to structurally and functionally regenerate damaged tissues, which requires the formation of new blood vessels that supply oxygen and nutrients by the process of angiogenesis. Stem cells are a promising tool in regenerative medicine due to their combined differentiation and paracrine angiogenic capacities. The study of their proangiogenic properties and associated potential for tissue regeneration requires complex in vivo models comprising all steps of the angiogenic process. The highly vascularized extraembryonic chorioallantoic membrane (CAM) of fertilized chicken eggs offers a simple, easy accessible, and cheap angiogenic screening tool compared to other animal models. Although the CAM assay was initially primarily performed for evaluation of tumor growth and metastasis, stem cell studies using this model are increasing. In this review, a detailed summary of angiogenic observations of different mesenchymal, cardiac, and endothelial stem cell types and derivatives in the CAM model is presented. Moreover, we focus on the variation in experimental setup, including the benefits and limitations of in ovo and ex ovo protocols, diverse biological and synthetic scaffolds, imaging techniques, and outcome measures of neovascularization. Finally, advantages and disadvantages of the CAM assay as a model for angiogenesis in tissue engineering in comparison with alternative in vivo animal models are described

Damage to Ventromedial Prefrontal Cortex Impairs Judgment of Harmful Intent

SummaryMoral judgments, whether delivered in ordinary experience or in the courtroom, depend on our ability to infer intentions. We forgive unintentional or accidental harms and condemn failed attempts to harm. Prior work demonstrates that patients with damage to the ventromedial prefrontal cortex (VMPC) deliver abnormal judgments in response to moral dilemmas and that these patients are especially impaired in triggering emotional responses to inferred or abstract events (e.g., intentions), as opposed to real or actual outcomes. We therefore predicted that VMPC patients would deliver abnormal moral judgments of harmful intentions in the absence of harmful outcomes, as in failed attempts to harm. This prediction was confirmed in the current study: VMPC patients judged attempted harms, including attempted murder, as more morally permissible relative to controls. These results highlight the critical role of the VMPC in processing harmful intent for moral judgment

RflM mediates target specificity of the RcsCDB phosphorelay system for transcriptional repression of flagellar synthesis in Salmonella enterica: Repression of flhDC transcription by a RcsB-RflM complex

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post- transcriptional levels. The flagellar master regulator FlhD₄C₂ resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD₄C₂- dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM- dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis

High peak-power stretcher-free femtosecond fiber amplifier using passive spatio-temporal coherent combining

International audienceWe report on the passive coherent combining of up to 8 temporally and spatially separated ultrashort pulses amplified in a stretcher-free ytterbium-doped fiber system. An initial femtosecond pulse is split into 4 temporal replicas using divided-pulse amplification, and subsequently divided in two counter-propagating beams in a Sagnac interferometer containing a fiber amplifier. The spatio-temporal distribution of the peak-power inside the amplifier allows the generation of record 3.1 µJ and 50 fs pulses at 1 MHz of repetition rate with 52 MW of peak-power from a stretcher-free fiber amplifier and without additional nonlinear post-compression stages

Compact, simple and robust cross polarized wave generation source of few-cycle, high-contrast pulses for seeding petawatt-class laser systems

International audienceA compact and robust, dual-crystal cross polarized wave generation setup combined with a hollow waveguide filter is implemented to deliver few-cycle, high-contrast laser pulses sourced from a commercial multipass Ti:Sa amplifier. The initial 25-fs pulses with a temporal contrast of 108 are shortened to 10 fs with an improved contrast of at least 10^10. The single nonlinear stage for spectral broadening and contrast enhancement of a commercial amplifier serves as an ideal injector for petawatt-class laser systems