Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues

<p>Abstract</p> <p>Background</p> <p>Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression.</p> <p>Results</p> <p>We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons.</p> <p>Conclusion</p> <p>We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.</p

Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9

AbstractBone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that play essential roles in human folliculogenesis and ovulation. Their bioactivity is tightly regulated through phosphorylation, likely to occur within the Golgi apparatus of the secretory pathway. Here we show that Golgi apparatus casein kinase (G-CK) catalyzes the phosphorylation of rhBMP-15 and rhGDF-9. rhBMP-15, in particular, is an excellent substrate for G-CK. In each protein a single residue is phosphorylated by G-CK, corresponding to the serine residue at the sixth position of the mature region of both rhBMP-15 and rhGDF-9, whose phosphorylation is required for biological activity

Test-Driven Design of an Active Dual-Polarized Log-Periodic Antenna for the Square Kilometre Array

An active dual-polarized Log-Periodic antenna has been designed to meet the requirements of the low-frequency (50 - 350 MHz) radio telescope of the Square Kilometre Array (SKA). The integration of antenna and low noise amplifier has been conceived in order to achieve a high degree of testability. This aspect has been found to be crucial to obtain a smooth frequency response compatible with the SKA science cases. The design has also been driven by other factors such as the large-volume production (more than 130 000 antennas will be built) and the environmental conditions of the harsh Australian desert. A specific verification approach based on both wideband radiometric spectral and spatial measurements in relevant laboratory and in-situ conditions has been developed. Electromagnetic analyses and experimental results exhibit a very good agreement. In December 2019, this antenna was part of the reference solution for the System Critical Design Review of the SKA

Clinical utility of Next Generation Sequencing of plasma cell-free DNA for the molecular profiling of patients with NSCLC at diagnosis and disease progression

Background: The present study evaluates the utility of NGS analysis of circulating free DNA (cfDNA), which incorporates small amounts of tumor DNA (ctDNA), at diagnosis or at disease progression (PD) in NSCLC patients. Methods: Comprehensive genomic profiling on cfDNA by NGS were performed in NSCLC patients at diagnosis (if tissue was unavailable/insufficient) or at PD to investigate potential druggable molecular aberrations. Blood samples were collected as routinary diagnostic procedures, DNA was extracted, and the NextSeq 550 Illumina platform was used to run the Roche Avenio ctDNA Expanded Kit for molecular analyses. Gene variants were classified accordingly to the ESCAT score. Results: A total of 106 patients were included in this study; 44&nbsp;% of cases were requested because of tissue unavailability at the diagnosis and 56&nbsp;% were requested at the PD. At least one driver alteration was observed in 62&nbsp;% of cases at diagnosis. Driver druggable variants classified as ESCAT level I were detected in 34&nbsp;% of patients, including ALK-EML4, ROS1-CD74, EGFR, BRAF, KRAS p.G12C, PI3KCA. In the PD group, most patients were EGFR-positive, progressing to a first line-therapy. Sixty-three percent of patients had at least one driver alteration detected in blood and 17&nbsp;% of patients had a known biological mechanism of resistance allowing further therapeutic decisions. Conclusions: The present study confirms the potential of liquid biopsy to detect tumour molecular heterogeneity in NSCLC patients at the diagnosis and at PD, demonstrating that a significant number of druggable mutations and mechanisms of resistance can be detected by NGS analysis on ctDNA

Test-Driven Design of an Active Dual-Polarized Log-Periodic Antenna for the Square Kilometre Array

An active dual-polarized Log-Periodic antenna has been designed to meet the requirements of the low-frequency (50-350 MHz) radio telescope of the Square Kilometre Array (SKA). The integration of antenna and low noise amplifier has been conceived in order to achieve a high degree of testability. This aspect has been found to be crucial to obtain a smooth frequency response compatible with the SKA science cases. The design has also been driven by other factors such as the large-volume production (more than 130 000 antennas will be built) and the environmental conditions of the harsh Australian desert. A specific verification approach based on both wideband radiometric spectral and spatial measurements in relevant laboratory and in-situ conditions has been developed. Electromagnetic analyses and experimental results exhibit a very good agreement. In December 2019, this antenna was part of the reference solution for the System Critical Design Review of the SKA

An essential role for decorin in bladder cancer invasiveness

Muscle-invasive forms of urothelial carcinomas are responsible for most mortality in bladder cancer. Finding new treatments for invasive bladder tumours requires adequate animal models to decipher the mechanisms of progression, in particular the way tumours interact with their microenvironment. Herein, using the murine bladder tumour cell line MB49 and its more aggressive variant MB49-I, we demonstrate that the adaptive immune system efficiently limits progression of MB49, whereas MB49-I has lost tumour antigens and is insensitive to adaptive immune responses. Furthermore, we unravel a parallel mechanism developed by MB49-I to subvert its environment: de novo secretion of the proteoglycan decorin. We show that decorin overexpression in the MB49/MB49-I model is required for efficient progression, by promoting angiogenesis and tumour cell invasiveness. Finally, we show that these results are relevant to muscle-invasive human bladder carcinomas, which overexpress decorin together with angiogenesis- and adhesion/migration-related genes, and that decorin overexpression in the human bladder carcinoma cell line TCCSUP is required for efficient invasiveness in vitro. We thus propose decorin as a new therapeutic target for these aggressive tumours.Fil: El Behi, Mohamed. Institute Curie; Francia. Centre de Recherche de I; Francia. Inserm; FranciaFil: Krumeich, Sophie. Institute Curie; Francia. Inserm; FranciaFil: Lodillinsky, Catalina. Institute Curie; Francia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kamoun, Aurélie. Institute Curie; FranciaFil: Tibaldi, Lorenzo. Institute Curie; Francia. Inserm; FranciaFil: Sugano, Gaël. Institute Curie; Francia. Inserm; FranciaFil: de Reynies, Aurélien. Ligue Nationale Contre le Cancer; FranciaFil: Chapeaublanc, Elodie. Institute Curie; Francia. Centre National de la Recherche Scientifique; FranciaFil: Laplanche, Agnès. Centre National de la Recherche Scientifique; Francia. Institut de Cancérologie Gustave Roussy; FranciaFil: Lebret, Thierry. Hôpital Foch. Service d; Francia. Université de Versailles; FranciaFil: Allory, Yves. Inserm; FranciaFil: Radvanyi, François. Institute Curie; Francia. Centre National de la Recherche Scientifique; FranciaFil: Lantz, Olivier. Institute Curie; Francia. Inserm; FranciaFil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bernard Pierrot, Isabelle. Institute Curie; Francia. Centre National de la Recherche Scientifique; FranciaFil: Théery, Clotilde. Institute Curie; Francia. Inserm; Franci

Pregnancy outcomes and cytomegalovirus DNAaemia in HIV infected pregnant women with CMV

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Rate , correlates and outcomes of repeat pregnancy in HIV-infected women

Objectives: The aim of the study was to assess the rate, determinants, and outcomes of repeat pregnancies in women with HIV infection. Methods: Data from a national study of pregnant women with HIV infection were used. Main outcomes were preterm delivery, low birth weight, CD4 cell count and HIV plasma viral load. Results: The rate of repeat pregnancy among 3007 women was 16.2%. Women with a repeat pregnancy were on average younger than those with a single pregnancy (median age 30 vs. 33 years, respectively), more recently diagnosed with HIV infection (median time since diagnosis 25 vs. 51 months, respectively), and more frequently of foreign origin [odds ratio (OR) 1.36; 95% confidence interval (CI) 1.10–1.68], diagnosed with HIV infection in the current pregnancy (OR: 1.69; 95% CI: 1.35–2.11), and at their first pregnancy (OR: 1.33; 95% CI: 1.06–1.66). In women with sequential pregnancies, compared with the first pregnancy, several outcomes showed a significant improvement in the second pregnancy, with a higher rate of antiretroviral treatment at conception (39.0 vs. 65.4%, respectively), better median maternal weight at the start of pregnancy (60 vs. 61 kg, respectively), a higher rate of end-of-pregnancy undetectable HIV RNA (60.7 vs. 71.6%, respectively), a higher median birth weight (2815 vs. 2885 g, respectively), lower rates of preterm delivery (23.0 vs. 17.7%, respectively) and of low birth weight (23.4 vs. 15.4%, respectively), and a higher median CD4 cell count (+47 cells/μL), with almost no clinical progression to Centers for Disease Control and Prevention stage C (CDC-C) HIV disease (0.3%). The second pregnancy was significantly more likely to end in voluntary termination than the first pregnancy (11.4 vs. 6.1%, respectively). Conclusions: Younger and foreign women were more likely to have a repeat pregnancy; in women with sequential pregnancies, the second pregnancy was characterized by a significant improvement in several outcomes, suggesting that women with HIV infection who desire multiple children may proceed safely and confidently with subsequent pregnancies