A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

High serum levels of Wnt antagonists are known to be involved in delayed bone defect healing. Pharmaceutically active implant materials that can modulate the micromilieu of bone defects with regard to Wnt antagonists are therefore considered promising to support defect regeneration. In this study, we show the versatility of a macromer based biomaterial platform to systematically optimize covalent surface decoration with high-sulfated glycosaminoglycans (sHA3) for efficient scavenging of Wnt antagonist sclerostin. Film surfaces representing scaffold implants were cross-copolymerized from three-armed biodegradable macromers and glycidylmethacrylate and covalently decorated with various polyetheramine linkers. The impact of linker properties (size, branching) and density on sHA3 functionalization efficiency and scavenging capacities for sclerostin was tested. The copolymerized 2D system allowed for finding an optimal, cytocompatible formulation for sHA3 functionalization. On these optimized sHA3 decorated films, we showed efficient scavenging of Wnt antagonists DKK1 and sclerostin, whereas Wnt agonist Wnt3a remained in the medium of differentiating SaOS-2 and hMSC. Consequently, qualitative and quantitative analysis of hydroxyapatite staining as a measure for osteogenic differentiation revealed superior mineralization on sHA3 materials. In conclusion, we showed how our versatile material platform enables us to efficiently scavenge and inactivate Wnt antagonists from the osteogenic micromilieu. We consider this a promising approach to reduce the negative effects of Wnt antagonists in regeneration of bone defects via sHA3 decorated macromer based macroporous implants. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical charac teristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect

Increased pore size of scaffolds improves coating efficiency with sulfated hyaluronan and mineralization capacity of osteoblasts

Background: Delayed bone regeneration of fractures in osteoporosis patients or of critical-size bone defects after tumor resection are a major medical and socio-economic challenge. Therefore, the development of more effective and osteoinductive biomaterials is crucial. Methods: We examined the osteogenic potential of macroporous scaffolds with varying pore sizes after biofunctionalization with a collagen/high-sulfated hyaluronan (sHA3) coating in vitro. The three-dimensional scaffolds were made up from a biodegradable three-armed lactic acid-based macromer (TriLA) by cross-polymerization. Templating with solid lipid particles that melt during fabrication generates a continuous pore network. Human mesenchymal stem cells (hMSC) cultivated on the functionalized scaffolds in vitro were investigated for cell viability, production of alkaline phosphatase (ALP) and bone matrix formation. Statistical analysis was performed using student's t-test or two-way ANOVA. Results: We succeeded in generating scaffolds that feature a significantly higher average pore size and a broader distribution of individual pore sizes (HiPo) by modifying composition and relative amount of lipid particles, macromer concentration and temperature for cross-polymerization during scaffold fabrication. Overall porosity was retained, while the scaffolds showed a 25% decrease in compressive modulus compared to the initial TriLA scaffolds with a lower pore size (LoPo). These HiPo scaffolds were more readily coated as shown by higher amounts of immobilized collagen (+ 44%) and sHA3 (+ 25%) compared to LoPo scaffolds. In vitro, culture of hMSCs on collagen and/or sHA3-coated HiPo scaffolds demonstrated unaltered cell viability. Furthermore, the production of ALP, an early marker of osteogenesis (+ 3-fold), and formation of new bone matrix (+ 2.5-fold) was enhanced by the functionalization with sHA3 of both scaffold types. Nevertheless, effects were more pronounced on HiPo scaffolds about 112%. Conclusion: In summary, we showed that the improvement of scaffold pore sizes enhanced the coating efficiency with collagen and sHA3, which had a significant positive effect on bone formation markers, underlining the promise of using this material approach for in vivo studies. © 2019 The Author(s)

A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif

[EN] Two small viral proteins (DGBp1 and DGBp2) have been proposed to act in a concerted manner to aid intra- and intercellular trafficking of carmoviruses though the distribution of functions and mode of action of each protein partner are not yet clear. Here we have confirmed the requirement of the DGBps of Pelargonium flower break virus (PFBV), p7 and p12, for pathogen movement Studies focused on p12 have shown that it associates to cellular membranes, which is in accordance to its hydrophobic profile and to that reported for several homologs. However, peculiarities that distinguish p12 from other DGBp52 have been found. Firstly, it contains a leucine zipper-like motif which is essential for virus infectivity in plants. Secondly, it has an unusually long and basic N-terminal region that confers RNA binding activity. The results suggest that PFBV p12 may differ mechanistically from related proteins and possible roles of PFBV DGBps are discussed. (C) 2011 Elsevier Inc. All rights reserved.We gratefully thank Dr. Vicente Pallas for critical reading of the manuscript and Dolores Arocas and Isabella Avellaneda for their technical assistance. This research was supported by grant BFU2006-11230 and BFU2009-11699 from the Ministerio Ciencia e Innovacion (MICINN, Spain) and by grants ACOM/2006/210 and ACOMP/2009/040 (Generalitat Valenciana, GV) to C. H. S. M.-T. was the recipient of a predoctoral fellowship from GV and of a predoctoral contract from MICINN.Martínez Turiño, S.; Hernandez Fort, C. (2011). A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif. Virology. 413(2):310-319. https://doi.org/10.1016/j.virol.2011.03.001S310319413

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer

Funder: National Health and Medical Research Council; doi: http://dx.doi.org/10.13039/501100000925Abstract: Background: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC

Use of a High-Fidelity Patient Simulator to Introduce an Evidence-Based Emergency Manual into Certified Registered Nurse Anesthetist Practice

Certified Registered Nurse Anesthetists (CRNAs) administer anesthesia care to thousands of patients each year. Despite increased anesthetic safety, low-frequency, high-risk perioperative critical events still occur. Although CRNAs have been expected to rely on memory alone to manage the spectrum of these critical events, the use of an emergency manual (EM) may improve CRNA performance. At one Army community hospital, recent experiences with similar critical events led to the development of the following question: Will the use of high-fidelity simulation training with the staff CRNAs on the use of EMs produce both increased performance in key tasks and positive satisfaction scores related to the use of the emergency manual during critical events now and in the future? Twenty staff CRNAs participated in high-fidelity simulated scenarios involving low-frequency, high-risk critical events before and after training on the use of an EM based on a change strategy developed by Goldhaber-Fiebert and Howard in 2012. Changes in performance after training were evaluated using a tool designed by Arriaga et al. Before EM training, CRNAs completed 46.05% of key tasks in the simulated scenario. After EM training, CRNAs completed up to 94.02% of key tasks in simulated scenarios. The observed increase in the completion of key tasks by CRNAs during simulated critical events after EM training and increase in satisfaction scores demonstrates how a well-constructed training program facilitates the implementation of an evidence-based EM into practice and can improve the quality and consistency of CRNA performance during low-frequency, high-risk perioperative events

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4R24C/R24C/TPras mice

Human melanoma susceptibility is often characterized by germline inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UVR dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0 to 57%. Here we report that activated Cdk4 cooperates with activated Hras to greatly increase susceptibility to melanoma in mice. While UVR treatment failed to induce melanomas in Cdk4R24C/R24C mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4R24C/R24C/TPras animals, compared to TPras alone. This increased penetrance was dependant on the threshold of Cdk4 activation, as Cdk4R24C/+/TPras animals did not show an increase in UVR-induced melanoma penetrance compared to TPras alone. In addition, Cdk4R24C/R24C/TPras mice invariably developed multiple primary lesions, which occurred rarely in TPras mice. These results indicate that germline defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4R24C/R24C/TPras tumors were comparable histopathologically, but the latter were larger and more aggressive, and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4R24C/R24C/TPras mice, but not TPras mice, readily metastasized to regional lymph nodes. Thus it appears that in the mouse, Hras activation initiates UVR-induced melanoma development, while the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing larger, more aggressive, metastatic tumors