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The combination of controlled radical polymerization (CRP) reactions and click chemistry offers high potential for the preparation of multifunctional polymers and significantly broadens the application scope of functional soft matter materials. In order to demonstrate the strategies as well as the potential of this methodology combination, examples for end-group and side-chain modification of polymers produced by CRP methods and the use of the resulting materials in functional polymer films are given. [ABSTRACT FROM AUTHOR]

Two phase separating block copolymers with photolabile protected amino groups in one block have been synthesized through RAFT polymerization followed by efficient click modification. Techniques like NMR, GPC, and DSC were applied for the characterization of these functional materials. The block copolymers were used for the preparation of thin films where they assemble to form distinct nanostructures as detected by AFM analysis. [ABSTRACT FROM AUTHOR]

Two phase separating block copolymers equipped with functional groups (acid and alkyne) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Thin films of these materials were prepared and examined with regard to surface morphology, surface composition, and film stability. Self-assembled structures with domain sizes of about 40 nm were detected through atomik force microscopy (AFM) analysis while X-ray photoelectron spectroscopy measurements revealed a balanced surface exposure of the two segregated phases. Thus, reactive groups being present in both phases are specifically provided within nanoscopic surface areas. The films showed good stability on exposure to various solvents but the self-organized surface patterns were only resistant toward ethanol. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011 [ABSTRACT FROM AUTHOR]

Abstract: Block copolymers consisting of a pentafluorostyrene (PFS) block and a hydrophilic block were synthesized by RAFT polymerisation. The hydrophilic blocks consist of methacrylate derivatives, 4-hydroxystyrene or 4-vinylpyridine monomers. The block copolymers were obtained with narrow molecular weight distributions and the molecular weights were in good agreement with the theoretical values. In addition, a model thiol was reacted with the PFS moieties of the block copolymers. This polymer–analogous reaction was performed under ambient conditions in high yields resulting quantitatively in para-substitution of the pentafluorophenyl rings. Finally, thin films consisting of block copolymers that showed strong phase-segregation behaviour and ordered nanostructured surfaces consisting of both blocks were obtained. [Copyright &y& Elsevier]

Simple Summary: Research in large animal models has been hampered by the complexity to introduce new gene alterations, but this has been simplified by the discovery of the CRISPR/Cas system. Here, we have cloned a Cas9 minipig to generate a porcine model for pre-clinical research. Six viable piglets were produced and backcrossed to Göttingen minipigs for two generations. Primary cells from different organs were isolated, and multiple gene alterations were performed by CRISPR in vitro. In vivo activation of the Cas9 expression was conducted by viral delivery of the FlpO expression to the skin. Overall, we successfully cloned a Cas9-expressing minipig and confirmed gene alterations introduced by the CRISPR/Cas system to porcine cells. The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research. [ABSTRACT FROM AUTHOR]

Simple Summary: Lung cancer is by far the leading cause of cancer induced mortality worldwide with a median five-year survival rate of 19 percent. Genome sequencing of lung cancer samples has revealed several key mutated genes, which could be implicated in lung cancer formation. This study applied a mouse model of lung cancer based on CRISPR/Cas9 technology to functionally address key regulators of the mTor pathway, STK11 and PTEN. Our study revealed that loss of Stk11 drives lung adenocarcinoma progression, whereas Pten is dispensable. These functional mouse studies reveal that loss of Pten is non-essential for lung adenocarcinoma, which is in agreement with the low mutation rates of PTEN in human adenocarcinoma. In contrast, loss of Stk11 drive tumor progression and is often found mutated in human samples of lung adenocarcinoma. This study focused on STK11, PTEN, KRAS, and TP53, which are often found to be mutated in lung cancer. We compared Stk11 and Pten implication in lung cancer in combination with loss of Trp53 and gain of function of Kras in a CRISPR/Cas9 mouse model. Mice with loss of Stk11, Trp53, and KrasG12D mutation (SKT) reached human endpoint at around four months post-initiation. In comparison, mice with loss of Pten, Trp53, and KrasG12D mutation (PKT) survived six months or longer post-initiation. Pathological examination revealed an increase in proliferation in SKT deficient lung epithelia compared to PKT. This difference was independent of Pten loss, indicating that loss of Pten is dispensable for cell proliferation in lung adenocarcinoma. Furthermore, tumors with loss of Stk11, Trp53, and KrasG12D mutation had a significantly higher progression rate, monitored by PET/MRI scanning, compared to mice with loss of Pten, Trp53, and KrasG12D mutation, revealing that mutations in Stk11 are essential for adenocarcinoma progression. Overall, by using the CRISPR/Cas9 mouse model of lung adenocarcinoma, we showed that mutations in Stk11 are a key driver, whereas loss of Pten is dispensable for adenocarcinoma progression. [ABSTRACT FROM AUTHOR]

The vascular endothelium is a crucial interface that controls the recruitment of circulating leukocytes. Based on the luminal expression of the ephrin-B2 ligand by endothelial cells (ECs) and the expression of EphB receptors (EphBRs) by circulating monocytes, we hypothesized that EphBR-ephrinB interactions are involved in monocyte adhesion. Adhesion experiments with monocytic cells were performed on ECs that overexpressed either full-length ephrin-B2 or cytoplasmically truncated ephrin-B2 (ΔC-ephrin-B2). Atomic force microscopy confirmed similar adhesive strengths of EphBR-expressing J774 cells to ECs that either overexpressed full-length ephrin-B2 or truncated ΔC-ephrin-B2 (1-minute interaction). Yet, adhesion experiments under static or flow conditions for 30 minutes demonstrated the preferential adhesion of monocytic cells to ECs that overexpressed full-length ephrin-B2 but not to ΔC-ephrin-B2 or to ECs that had been mock transduced. Adhesion was blocked by ephrin-B2-specific and EphBR-specific antibodies. Correspondingly, adhesion of EphB4-receptor-overexpressing monocytes to ephrin-B2-positive ECs was further augmented. Trafficking experiments of cell-surface molecules revealed that, prior to internalization, the resulting EphB4-receptor-ephrin-B2 complex translocated from the luminal surface to inter-endothelial junctions. Lastly, full-length ephrin-B2 in ECs was also involved in monocyte transmigration. Collectively, our study identifies a role of EphBR-ephrinB interactions as a new step in the cascade of events leading to monocyte adhesion and transmigration through the vascular endothelium. [ABSTRACT FROM AUTHOR]

Single-cell transcriptomics (scRNA-seq) has revolutionized the understanding of the spatial architecture of tissue structure and function. Advancing the "transcript-centric" view of scRNA-seq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyze post-translational modifications. Here, by combining spatial cell sorting with transcriptomics and quantitative proteomics/phosphoproteomics, we established the spatially resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signaling mechanisms. Phosphorylation of receptor tyrosine kinases was detected preferentially in the central vein area, resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation identified Tie receptor signaling as a selective and specific regulator of vascular Wnt activity orchestrating angiocrine signaling, thereby controlling hepatocyte function during liver regeneration. Taken together, the study has yielded fundamental insight into the spatial organization of liver endothelial cell signaling. Spatial sorting may be employed as a universally adaptable strategy for multiomic analyses of scRNA-seq-defined cellular (sub)-populations. [Display omitted] • ScRNA-seq-guided spatial sort enables multiomic dissection of the liver vasculature • Liver sinusoidal endothelial cells have a hybrid vascular-lymphatic phenotype • Tyrosine phosphorylation of endothelial cell molecules is enriched on central vein • Endothelial Tie1 shapes hepatic Wnt signal zonation and promotes liver regeneration Inverso, Shi et al. generate a multiomic encyclopedia of liver endothelial cells (L-ECs) with spatial resolution of transcriptome, proteome, and phosphoproteome. The study provides insight into liver vascular zonation and a template for scRNA-seq-data-guided spatial proteome and phosphoproteome analyses. [ABSTRACT FROM AUTHOR]