Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the ␤-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn 2؉ . In the presence of Zn 2؉ the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn 2؉ a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein.We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn 2؉ -dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen. Matrilin-3 is a heteromeric extracellular matrix (ECM)2 protein (50 kDa) that is primarily expressed in the cartilage growth plate (1, 2). Matrilin-3 has a modular structure comprising a von Willebrand factor A domain (A-domain), four epidermal growth factor-like domains, and a coiled-coil domain, which facilitates oligomerization (1-4). Mutations in the A-domain of matrilin-3 have been shown to result in some forms of multiple epiphyseal dysplasia (MED), an autosomal dominant skeletal dysplasia characterized by short-limb dwarfism and early onset osteoarthritis (5, 6). The majority of MATN3 mutations that cause MED are missense mutations found within the ␤-strands and are predicted to affect the folding of the A-domain (6, 7). Previous in vitro and in vivo studies have shown that mutant matrilin-3 protein is retained within the rough endoplasmic reticulum of cells and elicits an unfolded protein response (7-9). This essentially renders biochemical and biophysical analysis of the effects of the mutations very difficult, because it is not possible to isolate these recombinant mutant proteins under non-denaturing conditions. MED is genetically heterogeneous and can also result in mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A1, COL9A2, and COL9A3) (reviewed in Ref. 10). COMP is the fifth member of the thrombos...

DNA immobilization onto support surfaces is required in biotechnological applications such as microarrays and gene delivery. This important interfacial molecular process can be mediated from a preadsobred cationic polymer. There is, however, a lack of understanding over the control of the interfacial composition and structural distribution of the DNA immobilized. We have used a combined approach of spectroscopic ellipsometry (SE), dual polarization interferometry (DPI) and neutron reflection (NR) to determine the interfacial polymer adsorption and the subsequent DNA binding. Cationic diblock copolymers incorporating 30 phosphorylcholine (PC) groups and different diethylaminoethyl groups, referred to as MPC30-DEAn, were chosen because of their well-defined molecular architecture. While our studies revealed different effects of surface charge and hydrophobicity, the amount of copolymers adsorbed on both model surfaces showed a broad trend of increase with solution pH, indicating a strong effect arising from pH-dependent charge density on the copolymers. In contrast, the copolymer structure and solution concentration showed a weak effect under the conditions studied. The subsequent DNA binding at pH 7 showed that on both surfaces the amount of DNA immobilized followed an approximate 1:1 charge interaction for all different DNA samples studied, irrespective of single or double strand, or different DNA size, indicating the dominant effect of electrostatic interaction between the two species. Both DPI and NR revealed consistent thickness increase upon DNA binding. Furthermore, with increasing DNA size, the interfacial layer became much thicker, and charge interaction drove more extensive interfacial mixing between the two species. Our results show that the amount of DNA immobilized is controlled by the amount of cationic copolymer preadsorbed that is in turn controlled by the solution pH and surface chemistry but that is barely affected by the type and concentration of DNA or cationic copolymer.

This study reports the development and use of a novel thermoresponsive polymeric nanofilm for controlling cell adhesion and growth at 37 °C, and then cell detachment for cell recovery by subsequent temperature drop to the ambient temperature, without enzymatic cleavage or mechanical scraping. A copolymer, poly(N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-(trimethoxysilyl)propyl methacrylate) (abbreviated PNIPAAm copolymer), was synthesized by free radical polymerization. The thermoresponses of the copolymer in aqueous solution were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature. The DLS measurements revealed the lower critical solution temperature (LCST) at approximately 30 °C. The PNIPAAm film stability and robustness was provided through silyl cross-linking within the film and with the hydroxyl groups on the substrate surface. Film thickness, stability, and reversibility with respect to temperature switches were examined by spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and contact angle measurements. The results confirmed the high extent of thermosensitivity and structural restoration based on the alterations of film thickness and surface wettability. The effective control of adhesion, growth, and detachment of HeLa and HEK293 cells demonstrated the physical controllability and cellular compatibility of the copolymer nanofilms. These PNIPAAm copolymer nanofilms could open up a convenient interfacial mediation for cell film production and cell expansion by nonenzymatic and nonmechanical cell recovery.

Laser Induced Breakdown Spectroscopy (LIBS) has the potential to allow direct, standoff measurement of contaminants on nuclear plant. Here, LIBS is evaluated as an analytical tool for measurement of Sr and Cs contamination on type 304 stainless steel surfaces. Samples were reacted in model acidic (PUREX reprocessing) and alkaline (spent fuel ponds) Sr and Cs bearing liquors, with LIBS multi-pulse ablation also explored to measure contaminant penetration. The Sr II (407.77nm) and Cs I (894.35nm) emission lines could be separated from the bulk emission spectra, though only Sr could be reliably detected at surface loadings >0.5mgcm. Depth profiling showed decay of the Sr signal with time, but importantly, elemental analysis indicated that material expelled from LIBS craters is redistributed and may interfere in later laser shot analyses.

Graphical abstract