STAR:a simple TAL effector assembly reaction using isothermal assembly

Transcription activator-like effectors (TALEs) contain modular programmable DNA binding domains. Fusing TALEs with effector domains creates synthetic transcription factors (TALE-TFs) or nucleases (TALENs), enabling precise gene manipulations. The construction of TALEs remains challenging due to their repetitive sequences. Here we report a simple TALE assembly reaction (STAR) that enables individual laboratories to generate multiple TALEs in a facile manner. STAR uses an isothermal assembly (‘Gibson assembly’) that is labour- and cost-effective, accessible, rapid and scalable. A small 68-part fragment library is employed, and the specific TALE repeat sequence is generated within ~8 hours. Sequence-verified TALENs or TALE-TF plasmids targeting 17 bp target sequences can be produced within three days, without the need for stepwise intermediate plasmid production. We demonstrate the utility of STAR through production of functional TALE-TFs capable of activating human SOX2 expression. STAR addresses some of the shortcomings of existing Golden Gate or solid-phase assembly protocols and enables routine production of TALE-TFs that will complement emerging CRISPR/Cas9-based reagents across diverse applications in mammalian stem cell and synthetic biology

Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

<p>Abstract</p> <p>Background</p> <p>Insulin-like growth factor-I (IGF-I) exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex.</p> <p>Methods</p> <p>An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to <it>Aequorea victoria </it>enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65) was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test.</p> <p>Results</p> <p>IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65) to the cell nucleus.</p> <p>Conclusion</p> <p>These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory reactions in glial cells.</p

Ripretinib in gastrointestinal stromal tumor: the long-awaited step forward

Ripretinib; Sarcoma; Inhibidor de la tirosina cinasaRipretinib; Sarcoma; Inhibidor de la tirosina quinasaRipretinib; Sarcoma; Tyrosine kinase inhibitorGastrointestinal stromal tumor (GIST) represents a paradigm for clinically effective targeted inhibition of oncogenic driver mutations in cancer. Five drugs are currently positioned as the standard of care for the treatment of advanced or metastatic GIST patients. This is the result of continuous, deep understanding of KIT and PDGFRA GIST oncogenic drivers as well as the resistance mechanisms associated to tumor progression. However, the complexity of GIST molecular heterogeneity is an evolving field, and critical questions remain open. Specifically, the clinical benefit of approved and/or investigated targeted agents is strikingly modest at advanced stages of the disease when compared with the activity of first-line imatinib. Ripretinib is a novel switch-pocket inhibitor with broad activity against KIT and PDGFRA oncoproteins and has recently demonstrated antitumoral activity across phase I to phase III clinical trials. Therefore, ripretinib has emerged as a new standard of care for advanced, multi-resistant GIST patients. Based on this data, the Food and Drug Administration has granted in 2020 the approval of ripretinib for GIST patients after progression to imatinib, sunitinib and regorafenib. This, in turn, constitutes a major breakthrough in sarcoma drug development, as there have not been new treatment approvals in GIST for nearly a decade. Herein, we provide a critical review on the preclinical and clinical development of ripretinib in GIST. Furthermore, we seek to assess the biological and clinical impact of this new standard of care on the course of the disease, aiming to provide an insight on future treatments strategies for the next coming years.The authors received no financial support for the research, authorship, and/or publication of this article

Aportación metodológica para determinar el índice de calidad del medio ambiente urbano y edificado del Área Metropolitana de Barcelona

El presente trabajo está orientado hacia la determinación de una metodología que evalúa características socio-residenciales, diagnostica el estado de bienestar del medio ambiente urbano del Área Metropolitana de Barcelona (AMB), responde al estado en que se encuentran las viviendas pre-existentes al 2001. La investigación incorpora una aportación al enfoque de valoración inmobiliaria sustentada en técnicas de valores sociales como parte de un contexto integral. La orientación al desarrollo sostenible constituye dar un enfoque de valoración inmobiliaria, es decir, retomando el valor de uso como una combinación de valores urbanos y de satisfacción residencial en la base del análisis. La hipótesis de la investigación nace a partir de desarrollar una metodología sustentada en indicadores parciales (simples) idóneos para la obtención del Índice de Calidad Edificada del AMB obtenido por el método del indicador sintético de distancia DP2. El objetivo de la investigación es demostrar la validación del método del indicador sintético de distancia.This work is oriented towards the determination of a methodology to assess socio-residential characteristics, diagnoses the welfare state of the urban environment in the metropolitan area of Barcelona (AMB), responds to the state that are pre-existing dwellings to 2001 . The research incorporates a contribution to real estate valuation approach supported by techniques of social values as part of a comprehensive context. The sustainable development orientation is an approach to property valuation, returning the value in use as a combination of urban values and residential satisfaction in the analysis. The hypothesis of the research stems from developing a methodology supported by partial indicators (simple) suitable for obtaining Built Quality Index of AMB obtained by the method of the synthetic indicator DP2 away. The objective of this research is to demonstrate the validation of the method of the synthetic indicator away.Peer Reviewe

New evidence of the Green Tuff deposits and post-caldera, recent explosive volcanic activity at Pantelleria volcano (Sicily Channel, Italy) recorded in near-vent marine areas

The record of the explosive activity of Pantelleria volcano is well documented by several distal tephra collected in various areas in and around the Mediterranean, while few tephrochronological studies exist on proximal marine areas. In this paper, we investigate three selected coring sites from the northern shoulder of the Pantelleria graben, about 15–30 km from the island, in near-vent position with respect to that volcanic source. Our multiproxy analyses revealed nine tephra layers, totally composed of juvenile materials, mostly ash and pumice fragments, as well as a coarse-grained tephra deposit at the bottom of one of the cores. The major element composition of glass shards indicates a very homogeneous geochemical composition for the tephra layers, suggesting their primary origin. Using litho-stratigraphic and paleoenvironmental proxies, we associated the tephra layers to the latest (&lt;∼13.7 ka) explosive activity of Pantelleria. The coarse-grained deposit, instead, due to its distinctive composition is considered to represent the near-vent, marine equivalent of the distal Y-6 tephra layer, related to the ∼45 ka old co-ignimbrite fallout deposit of the Green Tuff event. The geochemical characterization of such deposit, mostly rhyolitic and partly trachytic, appears to enlarge the bimodal composition recorded in other distal tephra (Y-6) reported in the literature, highlighting the potential of near-vent record in providing additional information on the eruptive history

Excessive age-related decline in functional ovarian reserve in infertile women: prospective cohort of 15,500 women

Context: Whether infertile women exhibit accelerated ovarian aging and whether a low ovarian reserve is overrepresented in infertility populations is not known. Objective: To compare the age-related decline in antral follicle count (AFC), a biomarker of the ovarian reserve, in fertile and infertile women. Design: Cross-sectional data from a large prospective cohort study conducted from January 2013 to December 2014. Setting: Thirteen fertility centers across Spain. Patients or Other Participants: Consecutive women aged 18 to 45 years of age attending the fertility centers either seeking fertility treatment or as fertile women wishing to act as potential oocyte donors. Intervention(s): Standardized AFC assessment on day 2 to 4 of the cycle. Main Outcome Measure(s): Age-related decline of AFC for both fertile and infertile women. Results: A total of 15 500 women, of whom 5722 were potential donors and 9778 were patients seeking fertility treatment, participated in the study. Average AFC was greater in potential oocyte donors than in infertile women (20 [interquartile range, 16–24] vs 10 [interquartile range, 6–15], respectively; P &lt; .001), a difference that was maintained after adjustment for age (P &lt; .001) in a model predicting log(AFC) from donor vs infertility, adjusting for 2-year age bands. The age-related decline in AFC was much steeper in infertile women compared with that of potential oocyte donors, with an increased prevalence of a low ovarian reserve (AFC &lt; 5) at all ages in infertile women. Conclusions: The age-related decline in AFC was substantially greater in infertility patients than potential oocyte donors. Overrepresentation in infertility populations of women with low ovarian reserve may be an additional functional cause of infertility

Epitaxially strained [001]-(PbTiO3_3)1_1(PbZrO3_3)1_1 superlattice and PbTiO3_3 from first principles

The effect of layer-by-layer heterostructuring and epitaxial strain on lattice instabilities and related ferroelectric properties is investigated from first principles for the [001]-(PbTiO$_3$)$_1$(PbZrO$_3$)$_1$ superlattice and pure PbTiO$_3$ on a cubic substrate. The results for the superlattice show an enhancement of the stability of the monoclinic r-phase with respect to pure PbTiO$_3$. Analysis of the lattice instabilities of the relaxed centrosymmetric reference structure computed within density functional perturbation theory suggests that this results from the presence of two unstable zone-center modes, one confined in the PbTiO$_3$ layer and one in the PbZrO$_3$ layer, which produce in-plane and normal components of the polarization, respectively. The zero-temperature dielectric response is computed and shown to be enhanced not only near the phase boundaries, but throughout the r-phase. Analysis of the analogous calculation for pure PbTiO$_3$ is consistent with this interpretation, and suggests useful approaches to engineering the dielectric properties of artificially structured perovskite oxides.Comment: 8 pages, 5 figure

New membranes obtained by grafted irradiated PVDF foils

The present work describes a new method to produce membranes of poly(Acrylicacid-Xmonomer) using the grafting procedure. PVDF foils irradiated with Ar+ beamwith energies between 30 and 150 keV were employed as substratum. Differentcombinations of monomers in water solutions were used: acrylic acid (AAc); acrylicacid - glycidyl methacrylate (AAc-GMA); acrylic acid - styrene (AAc-S), acrylic acid-N-isopropyl acrylamide (AAc-NIPAAm) and acrylic acid - N-isopropyl acrylamide -glycidyl methacrylate (AAc-NIPAAm-GMA). A large percentage of grafting results forspecific values of: ion fluence and energy, AAc and sulfuric acid concentration, anddifferent substrata PVDF polymorphous (alpha or beta). At a particular time of thegrafting process, the Poly(AAc-Xmonomer) membranes detach from the substratum andcontinue their grafting in the solution. This method is useful to produce increasedreplicated membranes of the irradiated original surface.Fil: Mazzei, R.. Universidad Tecnológica Nacional; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Garcia Bermudez, Gerardo Jose. Comisión Nacional de Energía Atómica; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Camporotondi, Daniela Edhit. Comisión Nacional de Energía Atómica. Gerencia del Área de Investigaciones y Aplicaciones no Nucleares. Gerencia de Física (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arbeitman, Claudia Roxana. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: del Grosso, Mariela Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica; Argentina. Universidad Tecnológica Nacional. Facultad Regional General Pacheco; ArgentinaFil: Behar, M.. Universidade Federal do Rio Grande do Sul; Brasi

Activated-Carbon-Doped Non-Solvent-Induced Phase-Inversion Membranes : A Comprehensive Study on Synthesis, Characterisation, and Performance Evaluation

Acknowledgments: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. For the purpose of open access, the author has applied a Creative Commons Attribution (CC-BY) licence to any Author-Accepted Manuscript version arising from this submission For the purpose of open access, the author has applied a Creative Commons Attribution (CC-BY) licence to any Author Accepted Manuscript version arising from this submission.Peer reviewedPublisher PD

The function of FGF signaling in the lens placode

AbstractPrevious studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2CKO placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction