Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-β, 1–14 days) and thyroid hormone hypertrophic medium (T3, 15–28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-β2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs’ therapeutic effect on cartilage regeneration and hypertrophy suppression.     

Chitosan Microspheres as Carriers for pH‐Indicating Species in Corrosion Sensing

Chitosan microspheres containing bromocresol green, cresol red, and phenolphthalein for corrosion detection, through pH change, are synthesized in order to be used in protective coatings for aluminium alloys. Microspheres containing corrosion detection species are characterized morphologically (SEM) and physico‐chemically (FTIR, TGA). Release studies (UV–vis) are performed in corrosion‐promoting conditions (pH, NaCl), and detection studies by immersion in media associated with corrosion activity while microspheres' sensing activity is evaluated visually. Electrochemical characterization of AA2024 substrates in the presence of chitosan spheres is performed to understand material performance, and a color change is observed as a result of local pH increase in cathodic areas when corrosion takes place. These findings can be correlated with the results from release studies and seem a promising approach for corrosion sensing purposes, not only because pH increase is possible to detect due to corrosion, but also because chitosan is considered an environmentally friendly material.     

GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.     

Phase-Field Modeling of Nugget Zone for a AZ31-Mg-Alloy Friction Stir Weld

This work presents simulation of microstructure evolution in the nugget zone (NZ) of a AZ31-Mg-alloy friction stir weld. The process parameters (tool geometrical characteristics, rotational speed, travel speed, applied load) have been correlated with the resulting microstructural features in the NZ of the weld (grain size and population) with the aid of the MICRESS software, which provides the ability to simulate both nucleation and grain growth during dynamic recrystallization phenomena evolving in the NZ during the weld thermal cycle. The input parameters of the developed model include the tool geometry, the welding conditions as well as the recrystallization energy, the grain boundary mobility and specific material properties. NZ microstructure obtained by simulation shows good agreement with experimental measurements for both grain population and size.     

Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A₁, C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A₁- and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A₁-ring phenol and H-ring C-4’ hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.     

Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of pre‐mRNA splicing. Similar to other classes of nucleic acid‐based therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinyl‐tetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strain‐promoted azide‐alkyne cycloaddition conjugation with splice‐switching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest splice‐switching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2K‐mdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant splice‐switching at very low nanomolar concentrations. The presented aminoethylene‐lipopeptides are thus a promising platform for the generation of PMO‐therapeutics with a favorable activity/toxicity profile.     

Improved method for roadside barrier length of need modeling using real-world trajectories

The 2011 AASHTO Roadside Design Guide (RDG) contains perhaps the most widely used procedure for choosing an appropriate length of need (LON) for roadside barriers. However, this procedure has several limitations. The procedure uses a highly simplified model of vehicle departure, and the procedure does not allow designers to specify an explicit level of protection. A new procedure for choosing LON that addresses these limitations is presented in this paper. This new procedure is based on recent, real-world road departure trajectories and uses this departure data in a more realistic way. The new procedure also allows LON to be specified for a precisely known level of protection – a level which can be based on number of crashes, injury outcomes or even estimated crash cost – while still remaining straightforward and quick to use like the 2011 RDG procedure.