Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.     

Delayed self-regulation and time-dependent chemical drive leads to novel states in epigenetic landscapes

The epigenetic pathway of a cell as it differentiates from a stem cell state to a mature lineage-committed one has been historically understood in terms of Waddington''s landscape, consisting of hills and valleys. The smooth top and valley-strewn bottom of the hill represent their undifferentiated and differentiated states, respectively. Although mathematical ideas rooted in nonlinear dynamics and bifurcation theory have been used to quantify this picture, the importance of time delays arising from multistep chemical reactions or cellular shape transformations have been ignored so far. We argue that this feature is crucial in understanding cell differentiation and explore the role of time delay in a model of a single-gene regulatory circuit. We show that the interplay of time-dependent drive and delay introduces a new regime where the system shows sustained oscillations between the two admissible steady states. We interpret these results in the light of recent perplexing experiments on inducing the pluripotent state in mouse somatic cells. We also comment on how such an oscillatory state can provide a framework for understanding more general feedback circuits in cell development.     

Preparation and characterization of polyelectrolyte complex of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-trimethyl chitosan/gellan gum: evaluation for controlled release of ketoprofen

N,N,N-Trimethyl chitosan, a highly water soluble derivative of chitosan, has been made by reductive methylation of chitosan by a three-step process reported in literature. A novel polyelectrolyte complex of this derivative with gellan gum has been made by mixing the aqueous solutions of the two polymers. The complex was characterized by FTIR, TGA, DSC and SEM techniques. Maximum yield of the complex was obtained at pH 2.0 with a gellan gum:trimethylchitosan ratio of 3:1. The swelling study indicated pH responsiveness of the polyelectrolyte complex sample, with higher swelling under neutral or slightly basic conditions. In vitro studies on the release of the drug ketoprofen from the polyelectrolyte complex matrix were conducted in simulated gastric and intestinal fluids. The results indicated release of 85–90 % of the entrapped drug in the media of pH 6.8 and 7.4 and less than 7 % release in the medium of pH 1.2. The kinetic analysis indicated the drug release to be a first-order process. The mechanism of water transport and drug diffusion is shown to be of Fickian type. The results prove the suitability of the polyelectrolyte complex as a matrix material for delivery of drugs with short half life such as ketoprofen in the slow release mode.     

Effect of Alumina Reactivity on the Densification and Properties of Al2O3–Cr2O3 Refractories

Alumina‐chrome (Al₂O₃–Cr₂O₃) refractories with Al₂O₃:Cr₂O₃ molar ratio 1:1 were synthesized in the temperature range of 1400–1700°C by conventional solid–oxide reaction route. The effect of different aluminas (viz., hydrated and calcined) on the densification, microstructure, and properties of Al₂O₃–Cr₂O₃ refractories was investigated without changing the Cr₂O₃ source. The starting materials were analyzed to determine the chemical composition, mineralogy, density, surface area, and particle size. Sintered materials were characterized in terms of densification, phase assemblage, and mechanical strength at room temperature and at higher temperatures. Microstructural evolution at different sintering temperature was correlated with sintering characteristics. It can be concluded that the Al₂O₃–Cr₂O₃ refractories prepared with hydrated alumina as Al₂O₃ source show better densification and hot mechanical strength than corresponding calcined variety.     

Crystallography of solid–liquid interface and evolution of texture during directional solidification of Tb0.3Dy0.7Fe1.95 alloy

The effect of growth texture on the magnetostriction of ternary Tb_0.3Dy_0.7Fe_1.95 was studied by conducting unidirectional solidification experiments using a zoning set-up. Detailed texture evolutions were studied using X-ray diffraction on samples obtained by varying growth rates from 18 to 72 cm/h, under a temperature gradient of 100 °C/cm. The estimated texture co-efficient and pole figures of the samples indicate that during the onset of the solidification, <110> and <331>/‘rotated <110>’ texture components nucleate and grow in all the samples. However, as the solidification progresses, <112> texture component becomes dominant at higher growth rate. This results in an improvement of magnetostriction from 1000 to 1300 microstrains for samples grown at growth rates of 18 and 72 cm/h respectively. The transition of preferred growth direction occurs through intermediate orientations <123>. An attempt has been made in this paper to explain the occurrence of different growth texture by considering the stability of growing interface, its planar packing fraction and atomic stacking sequence of several low index planes.     

A new ternary phase in the system CaO–Al2O3–Cr2O3: Crystal structure and thermal expansion of CaAl2Cr2O7

Polycrystalline material of a novel phase in the system CaO–Al₂O₃–Cr₂O₃ has been obtained by solid-state reactions. Chemical analysis indicated the composition CaAl₂Cr₂O₇. Single-crystal growth of the new compound using borax as a mineralizer was successful. Diffraction experiments at ambient conditions on a crystal with composition CaAl_2.13Cr_1.87O₇ yielded the following basic crystallographic data: space group P 3, a = 7.7690(5) Å, c = 7.6463(5) Å, V = 399.68(6) ų, Z = 3. Structure determination and subsequent least-squares refinements resulted in a residual of R(|F|) = 2.3% for 1440 independent observed reflections and 113 parameters. To the best of our knowledge, the structure of CaAl_2.13Cr_1.87O₇ or CaAl₂Cr₂O₇ represents a new structure type. It belongs to the group of double layer structures where individual double layers contain octahedrally and tetrahedrally coordinated cation positions. Linkage between neighboring sheet packages is provided by additional calcium cations. Furthermore, thermal expansion has been studied in the interval between 29 and 790°C using in situ high-temperature single-crystal diffraction. No indications for a structural phase transition were observed. From the evolution of the lattice parameters the thermal expansion tensor has been obtained. A pronounced anisotropy is evident. The response of structural building units to variable temperature has been discussed.     

Cracking Characteristics of RC Beams Strengthened with FRP System

The cracking characteristics of fiber-reinforced polymer (FRP) strengthened reinforced concrete (RC) beams in both the short- and long-term is addressed in this paper. First, an empirical equation based on regression analysis of test results obtained from 36 beams was derived for the evaluation of crack widths in FRP-strengthened RC beams under short-term loading. The equation accounts for the effective concrete area in tension, steel stress, proximity of tensile longitudinal reinforcement, and primary crack height. Next, the long-term crack widths of glass FRP-strengthened RC beams under sustained loads were studied. Beams strengthened with glass FRP laminates showed improved cracking characteristics with smaller crack widths compared to conventional RC beams. Based on the investigation, two empirical equations are presented to compute the long-term crack widths in FRP-strengthened beams.     

Phase relationship,magnetic properties and Mössbauer studies in as cast and directionally solidified Tb0.3Dy0.7Fe1.95

Tb_0.3Dy_0.7Fe_1.95 alloy was cast and directionally solidified by modified Bridgman technique at different solidification rates and investigated for microstructural features and magnetic properties. The phase relationship between the co-existing phases viz., (Tb,Dy)Fe₂, (Tb,Dy)Fe₃ and (Tb,Dy)-rich are studied as a function of solidification rate to evolve a structure–property correlation. Higher solidification rate improves <110> grain texture and favour large magnetostriction. The magnetization and Mössbauer studies indicate no perceptible change in Fe magnetic moment, with a slight deviation in the 1:3 intensity ratio of the two sextets. Comparing the Mössbauer and microstructural results a relationship has been established between the intensity ratio and the volume fraction of the deleterious minor phase (Tb,Dy)Fe₃ in (Tb,Dy)Fe₂ microstructure.     

Human SMILE-Derived Stromal Lenticule Scaffold for Regenerative Therapy: Review and Perspectives

A transparent cornea is paramount for vision. Corneal opacity is one of the leading causes of blindness. Although conventional corneal transplantation has been successful in recovering patients’ vision, the outcomes are challenged by a global lack of donor tissue availability. Bioengineered corneal tissues are gaining momentum as a new source for corneal wound healing and scar management. Extracellular matrix (ECM)-scaffold-based engineering offers a new perspective on corneal regenerative medicine. Ultrathin stromal laminar tissues obtained from lenticule-based refractive correction procedures, such as SMall Incision Lenticule Extraction (SMILE), are an accessible and novel source of collagen-rich ECM scaffolds with high mechanical strength, biocompatibility, and transparency. After customization (including decellularization), these lenticules can serve as an acellular scaffold niche to repopulate cells, including stromal keratocytes and stem cells, with functional phenotypes. The intrastromal transplantation of these cell/tissue composites can regenerate native-like corneal stromal tissue and restore corneal transparency. This review highlights the current status of ECM-scaffold-based engineering with cells, along with the development of drug and growth factor delivery systems, and elucidates the potential uses of stromal lenticule scaffolds in regenerative therapeutics.     

A new route of cross-linking of carboxylated acrylonitrile-butadiene rubber via zinc oxide-amino acid network formation

Herein, we present a strategy that leads to the formation of a high-performance elastomeric material based on the carboxylated acrylonitrile-butadiene rubber (XNBR). A unique cross-linking route for XNBR has been designed, which does not involve typical cross-linking agents such as sulfur. In this study, the show cause-effect of zwitterionic compounds as the secondary cross-linking agent for ionic elastomer has been investigated. Naturally occurring lysine (Ly) and tryptophan (Trp) amino acid are the zwitterionic compounds, which have been explored here. This approach results in the formation of a cross-linked ionic elastomer that houses a dual ionic network structure formed by zinc oxide (ZnO) and amino acid. XNBR cross-linked by ZnO and different amino acid compounds exhibit superior physical properties as compared to the conventional cross-linking system. Fourier transform infrared spectroscopy, transmission electron microscope, and swelling study analyzes confirm the existence of zinc-carboxylate and zwitterionic network in XNBR after the cross-linking process. The dynamic mechanical analysis, differential scanning calorimetry, and oscillating disk rheometer analyzes show various thermal relaxations and transitions present in the dual cross-linked XNBR. The ZnO and amino acid cross-linked XNBR rubber with 2.5 phr of Ly and 5 phr of Trp show the highest tensile strength of 42.4 and 39.7 MPa, respectively, which is mostly higher than the previously reported values for ionic cross-linked elastomers.