Hyperbranched, Functional Polyethoxysiloxanes: Tunable Molecular Building Blocks.

Functional silanes are multifaceted cross-linkers, compatibilizers, coupling agents, and surface modifiers. Herein, we present organofunctional polysiloxane building blocks that offer great versatility in terms of molecular weight, degree of condensation, and the choice and loading of organic substituent groups. The organofunctional polyethoxysilanes (funPEOS) are prepared in a one-pot, two-step process: synthesis of the PEOS carrier/substrate, followed by grafting a functional silane "shell", both based on condensation with acetic anhydride. The reaction was optimized at the lab scale and scaled up to a 7 L reactor. The acetylation, condensation, and hyperbranched structure of the carrier were confirmed by 29Si NMR, while 29Si-29Si 2D INADEQUATE NMR provides strong evidence for the grafting of functional silanes onto the carrier (Q-T coupling). IR, 1H, and 13C NMR spectroscopy demonstrate that the functional groups remain intact. The molar mass can be tailored by stoichiometric control of the acetic anhydride to silane monomer ratio (M n 3500-20,000 g/mol). The compounds are stable organic liquids with a long shelf life. Selected applications are presented: scratch-resistant coatings with water contact angles of ∼90°, stable water emulsions, and surfactant-free, mesoporous silica foams.

Porous and Ultra-Flexible Crosslinked MXene/Polyimide Composites for Multifunctional Electromagnetic Interference Shielding.

Lightweight, ultra-flexible, and robust crosslinked transition metal carbide (Ti3C2 MXene) coated polyimide (PI) (C-MXene@PI) porous composites are manufactured via a scalable dip-coating followed by chemical crosslinking approach. In addition to the hydrophobicity, anti-oxidation and extreme-temperature stability, efficient utilization of the intrinsic conductivity of MXene, the interfacial polarization between MXene and PI, and the micrometer-sized pores of the composite foams are achieved. Consequently, the composites show a satisfactory X-band electromagnetic interference (EMI) shielding effectiveness of 22.5 to 62.5 dB at a density of 28.7 to 48.7 mg cm-3, leading to an excellent surface-specific SE of 21,317 dB cm2 g-1. Moreover, the composite foams exhibit excellent electrothermal performance as flexible heaters in terms of a prominent, rapid reproducible, and stable electrothermal effect at low voltages and superior heat performance and more uniform heat distribution compared with the commercial heaters composed of alloy plates. Furthermore, the composite foams are well attached on a human body to check their electromechanical sensing performance, demonstrating the sensitive and reliable detection of human motions as wearable sensors. The excellent EMI shielding performance and multifunctionalities, along with the facile and easy-to-scalable manufacturing techniques, imply promising perspectives of the porous C-MXene@PI composites in next-generation flexible electronics, aerospace, and smart devices.

Implementing an intensified antibiotic stewardship programme targeting daptomycin use in orthopaedic surgery: a cost-benefit analysis from the hospital perspective.

BACKGROUND: Hospital antibiotic stewardship (ABS) programmes offer several evidence-based tools to control prescription rates of antibiotics in different settings, influence the incidence of nosocomial infections and to contain the development of multi-drug-resistant bacteria. In the context of endoprosthetic surgery, however, knowledge of core antibiotic stewardship strategies, comparisons of costs and benefits of hospital ABS programmes are still lacking. MATERIALS AND METHODS: We identified a high daptomycin use for the treatment of methicillin-sensitive staphylococcal infections as a potential target for our ABS intervention. In addition, we endorsed periprosthetic tissue cultures for the diagnosis of PJI. Monthly antibiotic use data were obtained from the hospital pharmacy and were expressed as WHO-ATC defined daily doses (DDD) and dose definitions adapted to local guidelines (recommended daily doses, RDD), normalized per 1000 patient days. The pre-intervention period was defined from February 2012 through January 2014 (24 months). The post-intervention period included monthly time points from February 2014 to April 2015 (15 months). For a basic cost-benefit analysis from the hospital perspective, three cost drivers were taken into account: (1) the cost savings due to changes in antimicrobial prescribing; (2) costs associated with the increase in the number of cultured tissue samples, and (3) the appointment of an infectious disease consultant. Interrupted time-series analysis (ITS) was applied. RESULTS: Descriptive analysis of the usage data showed a decline in overall use of anti-infective substances in the post-intervention period (334.9 vs. 221.4 RDDs/1000 patient days). The drug use density of daptomycin dropped by -75 % (51.7 vs. 12.9 RDD/1000 patient days), whereas the utilization of narrow-spectrum penicillins, in particular flucloxacillin, increased from 13.8 to 33.6 RDDs/1000 patient days. ITS analysis of the consumption dataset showed significant level changes for overall prescriptions, as well as for daptomycin (p < 0.001) and for narrow-spectrum penicillins (p = 0.001). The total costs of antibiotic consumption decreased by an estimated € 4563 per month (p < 0.001), and around 90 % of these savings were linked to a decrease in daptomycin consumption. Overall, the antibiotic stewardship programme was beneficial, as monthly cost savings of € 2575 (p = 0.005) were achieved. INTERPRETATION: In this example of large endoprosthetic surgery department in a community-based hospital, the applied hospital ABS programme targeting daptomycin use has shown to be feasible, effective and beneficial compared to no intervention.

Development and validation of an HPLC method to determine metabolites of 5-hydroxymethylfurfural (5-HMF).

The food component 5-hydroxymethylfurfural is supposed to have antioxidative properties and is therefore used as an acting agent in a novel anticancer infusion solution, named Karal®, and an oral supplementation. Previous studies showed that after oral and intravenous application, the substance is completely decomposed to its metabolites: 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine. The formation of a fourth metabolite, namely 5-sulphoxymethylfurfural, is still not clarified according to literature. Due to commercial unavailability, synthesis of 5-sulphoxymethylfurfural was conducted and a synthesis procedure for N-(hydroxymethyl)furoyl glycine had to be developed. Identification of the synthesised compounds was proven by LC-MS and NMR. An appropriate HPLC method was established to obtain good separation of the four possible metabolic substances and 5-hydroxymethylfurfural within 12 min via a HILIC column (150 × 4.6 mm, 5 µm) using a gradient grade system switching from mobile phase A (ACN/ammonium formate 100 mM, pH 2.35, 95:5, v/v) to mobile phase B (ACN/ammonium formate 100 mM, pH 2.35, 85:15, v/v). The procedure was afterward validated following ICH guidelines in terms of selectivity, linearity, precision, LOD, and LOQ.

GAS2L3, a target gene of the DREAM complex, is required for proper cytokinesis and genomic stability.

The mammalian DREAM complex is a key regulator of cell-cycle-regulated gene transcription and drives the expression of many gene products required for mitosis and cytokinesis. In this study, we characterized GAS2L3, which belongs to the GAS2 family of proteins with putative actin- and microtubule-binding domains as a target gene of DREAM. We found that GAS2L3 localizes to the spindle midzone and the midbody during anaphase and cytokinesis, respectively. Biochemical studies show that GAS2L3 binds to and bundles microtubules as well as F-actin in vitro. Strikingly, the RNAi-mediated knockdown of GAS2L3 results in chromosome segregation defects in multinucleated cells and in cells with multi-lobed nuclei. Likewise, chronic downregulation of GAS2L3 causes chromosome loss and aneuploidy. Time-lapse videomicroscopy experiments in GAS2L3-knockdown cells reveal abnormal oscillation of chromatin and the spindle during cytokinesis. Taken together, our data reveal novel, important roles of GAS2L3 for faithful cell division. Our work thus contributes to the understanding of how DREAM regulates cytokinesis.

An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC.

Lin9, a subunit of the mammalian DREAM complex, is essential for embryonic development, for survival of adult mice, and for tumor suppression.

The retinoblastoma tumor suppressor protein (pRB) and related p107 and p130 "pocket proteins" function together with the E2F transcription factors to repress gene expression during the cell cycle and development. Recent biochemical studies have identified the multisubunit DREAM pocket protein complexes in Drosophila melanogaster and Caenorhabditis elegans in regulating developmental gene repression. Although a conserved DREAM complex has also been identified in mammalian cells, its physiological function in vivo has not been determined. Here we addressed this question by targeting Lin9, a conserved core subunit of DREAM. We found that LIN9 is essential for early embryonic development and for viability of adult mice. Loss of Lin9 abolishes proliferation and leads to multiple defects in mitosis and cytokinesis because of its requirement for the expression of a large set of mitotic genes, such as Plk1, Aurora A, and Kif20a. While Lin9 heterozygous mice are healthy and normal, they are more susceptible to lung tumorigenesis induced by oncogenic c-Raf than wild-type mice. Together these experiments provide the first direct genetic evidence for the role of LIN9 in development and mitotic gene regulation and they suggest that it may function as a haploinsufficient tumor suppressor.

The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosis.

Regulated gene expression is critical for the proper timing of cell cycle transitions. Here we report that human LIN-9 has an important function in transcriptional regulation of G2/M genes. Depletion of LIN-9 by RNAi in human fibroblasts strongly impairs proliferation and delays progression from G2 to M. We identify a cluster of G2/M genes as direct targets of LIN-9. Activation of these genes is linked to an association between LIN-9 and B-MYB. Chromatin immunoprecipitation assays revealed binding of both LIN-9 and B-MYB to the promoters of G2/M regulated genes. Depletion of B-MYB recapitulated the biological outcome of LIN-9 knockdown, including impaired proliferation and reduced expression of G2/M genes. These data suggest a critical role for human LIN-9, together with B-MYB, in the activation of genes that are essential for progression into mitosis.

Silencing of the meiotic genes SMC1beta and STAG3 in somatic cells by E2F6.

E2F6, a member of the E2F-family of transcription factors, is a retinoblastoma protein-independent transcriptional repressor. E2F6 associates with polycomb group (Pc-G) multiprotein complexes that contain histone H3 methyltransferases, suggesting that E2F6 represses genes by covalent histone modification. However, genes that are repressed by E2F6 via a mechanism that involves histone H3 methylation have not been identified. Using cDNA microarray experiments comparing wild-type and E2f6-/- mouse embryonic fibroblasts, we now found that E2F6 is required to silence the meiosis-specific genes SMC1beta and STAG3 in somatic cells. Re-expression of E2F6 in E2f6-/- cells was sufficient to restore their repression. E2F6 binds in vivo to the promoters of these genes through a conserved binding site. Transcriptional repression of SMC1beta and STAG3 by E2F6 involves multiple mechanisms, including methylation of histone H3 on lysine 9 and lysine 27. Our findings suggest a molecular mechanism for the stable transcriptional silencing of meiotic genes in somatic cells by E2F6.

Inhibition of oncogenic transformation by mammalian Lin-9, a pRB-associated protein.

Genetic studies in Caenorhabditis elegans identified lin-9 to function together with the retinoblastoma homologue lin-35 in vulva differentiation. We have now identified a human homologue of Lin-9 (hLin-9) and provide evidence about its function in the mammalian pRB pathway. hLin-9 binds to pRB and cooperates with pRB in flat cell formation in Saos-2 cells. In addition, hLin-9 synergized with pRB and Cbfal to transactivate an osteoblast-specific reporter gene. In contrast, hLin-9 was not involved in pRB-mediated inhibition of cell cycle progression or repression of E2F-dependent transactivation. Consistent with these data, hLin-9 was able to associate with partially penetrant pRB mutants that do not bind to E2F, but retain the ability to activate transcription and to promote differentiation. hLin-9 can also inhibit oncogenic transformation, dependent on the presence of a functional pRB protein. RNAi-mediated knockdown of Lin-9 can substitute for the loss of pRB in transformation of human primary fibroblasts. These data suggest that hLin-9 has tumor-suppressing activities and that the ability of hLin-9 to inhibit transformation is mediated through its association with pRB.

Genetic analysis of adipogenesis through peroxisome proliferator-activated receptor gamma isoforms.

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that is a key regulator of adipogenesis and is present in two isoforms generated by alternative splicing, PPARgamma1 and PPARgamma2. Studies of the ability of each isoform to stimulate fat differentiation have yielded ambiguous results, in part because PPARgamma stimulates its own expression. We have thus undertaken a formal genetic analysis using PPARgamma-null fibroblast cell lines to assess the specific role of each individual isoform in adipogenesis. We show here that both PPARgamma1 and PPARgamma2 have the intrinsic ability to stimulate robust adipogenesis. Adipose cells stimulated by either PPARgamma1 or PPARgamma2 express a similar gene profile and show similar responses to insulin. However, in response to low ligand concentrations, PPARgamma2 shows a quantitatively greater ability to induce adipogenesis. Analyses involving coactivator binding and transcriptional assays indicate that PPARgamma2 has an enhanced ability to bind components of the DRIP/TRAP complex, coactivators required for fat differentiation.