Smart thermoresponsive coatings and surfaces for tissue engineering: switching cell-material boundaries

The smart thermoresponsive coatings and surfaces that have been explicitly designed for cell culture are mostly based on poly(N-isopropylacrylamide) (PNIPAAm). This polymer is characterized by a sudden precipitation on heating, switching from a hydrophilic to a hydrophobic state. Mammalian cells cultured on such thermoresponsive substrates can be recovered as confluent cell sheets, while keeping the newly deposited extracellular matrix intact, simply by lowering the temperature and thereby avoiding the use of deleterious proteases. Thermoresponsive materials and surfaces are powerful tools for creating tissue-like constructs that imitate native tissue geometry and mimic its spatial cellular organization. Here we review and compare the most representative methods of producing thermoresponsive substrates for cell sheet engineering.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.     

Analysis of single nucleotide polymorphisms at 13 loci within the transforming growth factor-induced factor gene shows no association with high myopia in Japanese subjects

A previous study in China first indicated that the transforming growth factor-induced factor (TGIF) is a probable candidate gene for high myopia. The purpose of our study was to investigate whether there are significant associations between high myopia and single nucleotide polymorphism (SNP) variants in the TGIF gene of Japanese subjects. Genomic DNA was collected from 330 Japanese subjects with high myopia and at a level refractive error was less than −9.25 Dsph and 330 randomized controls without high myopia. Thirteen SNPs were detected by polymerase chain reaction (PCR) and primer extension or by PCR and SNP-specific fluorogenic probes in all of the cases and controls. Thirteen SNPs were found within the TGIF genes of the cases and controls. Two of the SNPs were monomorphic and none of the 13 SNPs showed a significant result. The pairwise linkage disequilibrium (LD) mapping confirmed that these alleles have a comparatively strong LD index of >0.8 for D′ and >0.4 for r ². We found no statistical association between any of the 13 SNPs located on the TGIF gene and high myopia in Japanese subjects. Based on our study using Japanese subjects and the previous studies of TGIF gene polymorphism in Chinese and northern European subjects with myopia, there is no convincing evidence to prove a connection between nucleotide sequence variations in TGIF and high myopia.     

Studies on carbon material requirements for bacterial proliferation and spore germination under stress conditions: a new mechanism involving transmission of physical signals.

The growth of bacteria is often enhanced by addition of carbon materials such as graphite or activated charcoal to the growth medium. In this work, bacterial strains that strictly require such carbon materials under the ordinarily lethal stress caused by high concentrations of salt were isolated. The organisms were gram-positive, spore-forming, sugar-nonfermenting aerobic bacilli and were provisionally designated "Bacillus carbophilus" Kasumi after examination of their phenotypic traits. The growth- and germination-promoting effects of graphite and activated charcoal were demonstrated either quantitatively on agar plates containing fine crystals of the carbon materials mixed with a nonpermissive growth medium or qualitatively on agar plates on nonpermissive growth media half-covered with fine carbon particles. Further experiments demonstrated a novel feature of the phenomenon; i.e., the ability to induce colony formation on the nonpermissive plate was transmissible through the air, as well as through plastic or glass barriers. The mechanism probably involves transmission of physical signals regulating cell growth.     

Bioconversion of 3beta-hydroxy-5-cholenoic acid into chenodeoxycholic acid by rat brain enzyme systems

We have previously demonstrated that the rat brain contains three unconjugated bile acids, and chenodeoxycholic acid (CDCA) is the most abundantly present in a tight protein binding form. The ratio of CDCA to the other acids in rat brain tissue was significantly higher than the ratio in the peripheral blood, indicating a contribution from either a specific uptake mechanism or a biosynthetic pathway for CDCA in rat brain. In this study, we have demonstrated the existence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. To distinguish marked compounds from endogenous related compounds, 18O-labeled 3beta-hydroxy-5-cholenoic acid, 3beta,7alpha-dihydroxy-5-cholenoic acid, and 7alpha-hydroxy-3-oxo-4-cholenoic acid were synthesized as substrates for in vitro incubation studies. The results clearly suggest that 3beta-hydroxy-5-cholenoic acid was converted to 3beta,7alpha-dihydroxy-5-cholenoic acid by microsomal enzymes. The 7alpha-hydroxy-3-oxo-4-cholenoic acid was produced from 3beta,7alpha-dihydroxy-5-cholenoic acid by the action of microsomal enzymes, and Delta4-3-oxo acid was converted to CDCA by cytosolic enzymes. These findings indicate the presence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. Furthermore, this synthetic pathway for CDCA may relate to the function of 24S-hydroxycholesterol, which plays an important role in cholesterol homeostasis in the body.     

Effectiveness of using an unskilled model in action observation combined with motor imagery training for early motor learning in elderly people: a preliminary study

Abstract

Aim of the study: To investigate a more available model for the early phase of motor learning after action observation combined with motor imagery training in elderly people. To address the purpose, we focused on a slow, unskilled model demonstrating an occasional error.

Materials and methods: A total of 36 elderly people participated in the current study and were assigned to either the unskilled or skilled model observation groups (n?=?12, respectively), or the control group (n?=?12). The participants in the observation groups observed the assigned a video clip of an unskilled or skilled model demonstrating a ball rotation task. During the observation, the participants were instructed to imagine themselves as the person in the video clip. The participants in the control group read a scientific paper during the equivalent period of action observation and motor imagery. We measured ball rotation performance (the time required for five rotations, the number of ball drops) in pre- and post-intervention (observation combined with motor imagery training for intervention groups or reading for control group).

Results: Ball rotation performance (ball rotation speed) significantly improved in the unskilled model observation group compared to the other two groups.

Conclusions: Intervention for action observation using unskilled model combined with motor imagery was effective for improving motor performance during the early phase of motor learning.     

Poly(N-isopropylacrylamide) surface-grafted chitosan membranes as a new substrate for cell sheet engineering and manipulation

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosan membranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance ((1)H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously described methodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.     

Integrating elements and energy through the metabolic dependencies of gross growth efficiency and the threshold elemental ratio

Metabolic theory proposes that individual growth is governed through the mass‐ and temperature‐dependence of metabolism, and ecological stoichiometry posits that growth is maximized at consumer‐specific optima of resource elemental composition. A given consumer's optimum, the threshold elemental ratio (TER), is proportional to the ratio of its maximum elemental gross growth efficiencies (GGEs). GGE is defined by the ratio of metabolism‐dependent processes such that GGEs should be independent of body mass and temperature. Understanding the metabolic‐dependencies of GGEs and TERs may open the path towards a theoretical framework integrating the flow of energy and chemical elements through ecosystems. However, the mass and temperature scaling of GGEs and TERs have not been broadly evaluated. Here, we use data from 95 published studies to evaluate these metabolic‐dependencies for C, N and P from unicells to vertebrates. We show that maximum GGEs commonly decline as power functions of asymptotic body mass and exponential functions of temperature. The rates of change in maximum GGEs with mass and temperature are relatively slow, however, suggesting that metabolism may not causally influence maximum GGEs. We additionally derived the theoretical expectation that the TER for C:P should not vary with body mass and this was supported empirically. A strong linear relationship between carbon and nitrogen GGEs further suggests that variation in the TER for C:N should be due to variation in consumer C:N. In general we show that GGEs may scale with metabolic rate, but it is unclear if there is a causal link between metabolism and GGEs. Further integrating stoichiometry and metabolism will provide better understanding of the processes governing the flow of energy and elements from organisms to ecosystems.