Probabilistic failure analysis of bone using a finite element model of mineral–collagen composites

Microdamage accumulation is a major pathway for energy dissipation during the post-yield deformation of bone. In this study, a two-dimensional probabilistic finite element model of a mineral–collagen composite was developed to investigate the influence of the tissue and ultrastructural properties of bone on the evolution of microdamage from an initial defect in tension. The probabilistic failure analyses indicated that the microdamage progression would be along the plane of the initial defect when the debonding at mineral–collagen interfaces was either absent or limited in the vicinity of the defect. In this case, the formation of a linear microcrack would be facilitated. However, the microdamage progression would be scattered away from the initial defect plane if interfacial debonding takes place at a large scale. This would suggest the possible formation of diffuse damage. In addition to interfacial debonding, the sensitivity analyses indicated that the microdamage progression was also dependent on the other material and ultrastructural properties of bone. The intensity of stress concentration accompanied with microdamage progression was more sensitive to the elastic modulus of the mineral phase and the nonlinearity of the collagen phase, whereas the scattering of failure location was largely dependent on the mineral to collagen ratio and the nonlinearity of the collagen phase. The findings of this study may help understanding the post-yield behavior of bone at the ultrastructural level and shed light on the underlying mechanism of bone fractures.     

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent (ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg⁻¹ with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.     

Highly efficient method for 125I-radiolabeling of biomolecules using inverse-electron-demand Diels–Alder reaction

In this report, we present a rapid and highly efficient method for radioactive iodine labeling of trans-cyclooctene group conjugated biomolecules using inverse-electron-demand Diels–Alder reaction. Radioiodination reaction of the tetrazine structure was carried out using the stannylated precursor 2 to give ¹²⁵I-labeled product ([¹²⁵I]1) with high radiochemical yield (65 ± 8%) and radiochemical purity (>99%). For radiolabeling application of [¹²⁵I]1, trans-cyclooctene derived cRGD peptide and human serum albumin were prepared. These substrates were reacted with [¹²⁵I]1 under mild condition to provide the radiolabeled products [¹²⁵I]6 and [¹²⁵I]8, respectively, with excellent radiochemical yields. The biodistribution study of [¹²⁵I]8 in normal ICR mice showed significantly lower thyroid uptake values than that of ¹²⁵I-labeled human serum albumin prepared by a traditional radiolabeling method. Therefore [¹²⁵I]8 will be a useful radiolabeled tracer in various molecular imaging and biological studies. Those results clearly demonstrate that [¹²⁵I]1 will be used as a valuable prosthetic group for radiolabeling of biomolecules.     

Simple assay for adsorption of proteins to the air–water interface

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air–water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.     

Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose

Cellulase from Trichoderma reesei (Celluclast 1.5 L, Novozyme) was immobilized by sol–gel encapsulation, using binary or ternary mixtures of tetramethoxysilane (TMOS) with alkyl- or aryl-substituted trimethoxysilanes as precursors. Optimization of immobilization conditions resulted in 92 % recovery of total enzymatic activity in the best immobilized preparate. The immobilized cellulase exhibiting the highest activity, obtained from tetramethoxysilane and methyltrimethoxysilane precursors at 3:1 molar ratio, was investigated in the hydrolysis reaction of microcrystalline cellulose (Avicel PH101). Although the optimal values did not change significantly, both temperature and pH stabilities of the sol–gel entrapped cellulase improved compared to the native enzyme. Immobilization also conferred superior resistance against the inactivation effect of glucose. Reuse of the sol–gel entrapped cellulase showed 40 % retention of the initial activity after five batch hydrolysis cycles, demonstrating the potential of this biocatalyst for large-scale application.     

Characterization of Protein–Protein Interfaces

We analyze the characteristics of protein–protein interfaces using the largest datasets available from the Protein Data Bank (PDB). We start with a comparison of interfaces with protein cores and non-interface surfaces. The results show that interfaces differ from protein cores and non-interface surfaces in residue composition, sequence entropy, and secondary structure. Since interfaces, protein cores, and non-interface surfaces have different solvent accessibilities, it is important to investigate whether the observed differences are due to the differences in solvent accessibility or differences in functionality. We separate out the effect of solvent accessibility by comparing interfaces with a set of residues having the same solvent accessibility as the interfaces. This strategy reveals residue distribution propensities that are not observable by comparing interfaces with protein cores and non-interface surfaces. Our conclusions are that there are larger numbers of hydrophobic residues, particularly aromatic residues, in interfaces, and the interactions apparently favored in interfaces include the opposite charge pairs and hydrophobic pairs. Surprisingly, Pro-Trp pairs are over represented in interfaces, presumably because of favorable geometries. The analysis is repeated using three datasets having different constraints on sequence similarity and structure quality. Consistent results are obtained across these datasets. We have also investigated separately the characteristics of heteromeric interfaces and homomeric interfaces.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Synthesis and Characterization of Polysialic Acid–Uricase Conjugates for the Treatment of Hyperuricemia

Uricase, an enzyme used for the treatment of hyperuricemia, is conjugated with polysialic acid (PSA) of average molecular weight of 10 kDa by reductive amination in presence of NaCNBH3 in order to improve its pharmacological properties. Polysialylation with 50-,100-,150- and 200-fold molar excess of PSA increased the percentage substitution of the free amino groups on enzyme surface (46, 66, 78 and 80 % respectively). The SDS-PAGE is used to visualize the conjugates with increased molecular weight and it retained almost 65 % of their initial specific activity after conjugation. The stability studies at physiological condition reveals improved stability and activity than the native enzyme. The apparent KM of the enzyme has increased slightly from 4.18 × 10^?5 M to 5.46 × 10^?5 M suggesting that the affinity of the substrate to the enzyme has not been altered to a higher extent. The conjugates, when probed against anti-uricase antibodies generated in rabbit, showed a clean decline in the affinity by 35 % and also have retained double the catalytic activity than that of the native enzyme after exposure to antiserum. The results suggest that uricase-PSA conjugates can be used as an alternative to the conventional synthetic polymer-enzyme conjugates.     

Immobilization of lipase within carbon nanotube–silica composites for non-aqueous reaction systems

The immobilization of lipases within sol–gel derived silica, using multi-walled carbon nanotubes (MWNTs) as additives in order to protect the inactivation of lipase during sol–gel process and to enhance the stability of lipase, was investigated. Three sol–gel immobilized lipases (Candida rugosa, Candida antarctica type B, Thermomyces lanuginosus) with 0.33% (w/w) MWNT showed much higher activities than lipase immobilized without MWNT. The influence of MWNT content and MWNT shortened by acid treatment in the sol–gel process on the activity and stability of immobilized C. rugosa lipase was also studied. In hydrolysis reaction, immobilized lipase containing 1.1% pristine MWNT showed 7 times higher activity than lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT showed 10 times higher activity in esterification reaction, compared with lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT retained 96% of initial activity after 5 times reuse, while the lipase immobilized without MWNT was fully inactivated under the same condition.     

Reusable ω-transaminase sol–gel catalyst for the preparation of amine enantiomers

Heterogeneous ω-transaminase sol–gel catalysts were prepared and characterized in terms of immobilization degree, loading capacity and catalytic behavior in the kinetic resolution of racemic 1-phenylethylamine (a model compound) with sodium pyruvate in phosphate buffer (pH 7.5). The catalyst obtained when ω-transaminase from Arthrobacter sp. was encapsulated from the aqueous solution of the enzyme, isopropyl alcohol and polyvinyl alcohol in the sol–gel matrices, consisting of the 1:5 mixture of tetramethoxysilane and methyltrialkoxysilane, proved to be optimal including the reuse and storage stabilities of the catalyst. The optimized immobilizate was shown to perform well in the kinetic resolution of four structurally different aromatic primary amines in aqueous DMSO (10, v/v-%). The enzyme preparation showed synthetic potential by enabling the catalyst reuse in five consecutive preparative scale kinetic resolutions using 100 mM 1-phenylethylamine in aqueous DMSO (10, v/v-%). It was typical to fresh catalyst preparations that the kinetic resolution tended to exceed 50% before the reaction stopped leaving the (S)-amine unreacted while thereafter in reuse the reactions stopped at 50% conversion as expectable to highly enantioselective reactions.     

The requirement of zinc for improvement of crop yield and mineral nutrition in the maize–mungbean–rice system

A study was made over 3 years to find out an optimum rate of Zn application for the maize–mungbean–rice cropping system in a calcareous soil of Bangladesh. Zinc application was made at 0, 2 and 4 kg ha⁻¹ for maize (cv. Pacific 984, Thai hybrid) and at 0, 1 and 2 kg ha⁻¹ for rice (cv. BRRI dhan33), with no Zn application for mungbean (cv. BARI mung5). Effect of Zn was evaluated in terms of yield and mineral nutrients contents (N, P, S and Zn). All the three crops responded significantly to Zn application. The optimum rate of Zn for the maize–mungbean–rice cropping system was found to be 4–0–2 kg ha⁻¹ for the first year and 2–0–2 kg ha⁻¹ for subsequent years particularly when mungbean residue was removed, and such rates for mungbean residue incorporation being 4–0–1 and 2–0–1 kg ha⁻¹, respectively. For all crops, the Zn and N concentrations of grain were significantly increased with Zn application. For the case of grain-S, the concentration was significantly increased for maize and mungbean, but it remained unchanged for rice. The grain-P concentration on the other hand tended to decrease with Zn application. For maize, the grain-Zn concentration increased to 27.0 μg g⁻¹ due to 2 kg Zn ha⁻¹ treatment from 16.5 μg g⁻¹ for Zn control and at higher Zn rate (4 kg Zn ha⁻¹) the increment was very minimum. Another field experiment was performed over 3 years on the same soil to screen out maize varieties for Zn efficiency. Of the eight varieties tested, the BARI maize 6 and BARI hybrid maize 3 were found Zn in-responsive (Zn efficient) and the others Zn responsive (Zn-inefficient).     

Sponge spicules as blueprints for the biofabrication of inorganic–organic composites and biomaterials

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e., biosilicification) hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results.     

A carbon nanotube/silica sol–gel architecture for immobilization of horseradish peroxidase for electrochemical biosensor

A novel third-generation biosensor for hydrogen peroxide (H₂O₂) has been constructed based on horseradish peroxidase (HRP) immobilized by the sol–gel (SG) technology on carbon nanotube (CNT)-modified electrode. CNT has good promotion effects on the direct electron transfer between HRP and the electrode surface and the SG network provides a biocompatible microenvironment for enzyme. The immobilized HRP retained its bioelectrocatalytic activity for the reduction of hydrogen peroxide and can respond to the change of concentration of H₂O₂ rapidly. The heterogeneous electron transfer rate constant was evaluated to be 2.8 ± 0.4 s⁻¹. The amperometric response to H₂O₂ shows a linear relation in the range from 0.5 to 300 μmol l⁻¹ and a detection limit of 0.1 μmol l⁻¹ (S/N = 3). The K _M^app value of HRP immobilized on the electrode surface was found to be 1.35 mmol l⁻¹. The biosensor exhibited high sensitivity, rapid response and excellent long-term stability.     

Physicochemical Characterization of Efavirenz–Cyclodextrin Inclusion Complexes

Efavirenz (EFV) is an oral antihuman immunodeficiency virus type 1 drug with extremely poor aqueous solubility. Thus, its gastrointestinal absorption is limited by the dissolution rate of the drug. The objective of this study was to characterize the inclusion complexes of EFV with β-cyclodextrin (β-CD), hydroxypropyl β-CD (HPβCD), and randomly methylated β-CD (RMβCD) to improve the solubility and dissolution of EFV. The inclusion complexation of EFV with cyclodextrins in the liquid state was characterized by phase solubility studies. The solid-state characterization of various EFV and CD systems was performed by X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy analyses. Dissolution studies were carried out in distilled water using US Pharmacopeia dissolution rate testing equipment. Phase solubility studies provided an A_L-type solubility diagram for β-CD and A_P-type solubility diagram for HPβCD and RMβCD. The phase solubility data enabled calculating stability constants (K _s) for EFV-βCD, EFV-HPβCD, and EFV-RMβCD systems which were 288, 469, and 1,073 M⁻¹, respectively. The physical and kneaded mixtures of EFV with CDs generally provided higher dissolution of EFV as expected. The dissolution of EFV was substantially higher with HPβCD and RMβCD inclusion complexes prepared by the freeze drying method. Thus, complexation with HPβCD and RMβCD could possibly improve the dissolution rate-limited absorption of EFV.     

A two-dimensional gel electrophoretic procedure for the separation of complex mixtures of 4–12S RNAs

A two-dimensional gel electrophoretic method suitable for the separation of complex mixtures of RNA species in the size range of 4 to 12 S is described. A 3.6–11% polyacrylamide gradient gel containing a gradient of 0–7 m urea was used in the first dimension, and a transverse 3.6–22.6% polyacrylamide gradient gel containing 5 m urea was used in the second dimension. The method was applied to the separation of total cytoplasmic RNAs from a cellular slime mold. In this method reproducible fingerprints were obtained by the use of visible-marker RNA.     

Fractionation of functional polystyrenes,poly(ethylene oxide)s and poly(styrene)–b–poly(ethylene oxide) by liquid chromatography at the exclusion–adsorption transition point

The paper reports the fractionation of functional polystyrenes (PSs) and poly(ethylene oxide)s (PEOs) as well as their block copolymers, by liquid chromatography at the exclusion adsorption transition point (EATP–LC), also called “critical conditions” mode. In this specific elution mode (EATP–LC), the fractionation is only governed by the nature and the number of functions attached to the polymer backbone, independent of the molar mass distribution of the whole sample. Functional polystyrenes (α- and/or α,ω-alcohol-, acetal-, aldehyde- and acidic-PS) could be readily separated from non-functional polystyrenes under various chromatographic conditions. The technique also allowed the fractionation of poly(ethylene oxide)s and PS–PEO block copolymers. In the latter cases, moderately polar columns (grafted silica) and water-based polar eluents were required to obtain a satisfactory fractionation.     

Characterization of the Y-Chromosome Sardinian–Corsican Haplotype

The Y chromosome of 523 Italian male subjects was examined for the 49a,f TaqI haplotype XII and for two microsatellites, YCAIIa and YCAIIb. Results were then compared to other populations living in the western Mediterranean basin whom we had previously studied: 419 French (including 328 Corsicans), 46 Italians from Milan, and 73 Tunisians. Haplotype XII is present in 127 out of the 1061 examined samples (11.9%), and most of the haplotype XII subjects are of the compound haplotype YCAIIa-21 and YCAIIb-11. Two peaks of haplotype XII frequencies occur in the north of Sardinia (35%) and in the central area of Corsica (17.4%).     

Characterization of Wistar–Kyoto rats showing hyperadiponectinemia

AimsWistar–Kyoto rats (HA-WKY) kept in the author's laboratory showed higher levels of serum adiponectin (approximately 4-fold) compared with Wistar–Kyoto/Izm rats (WKY/Izm), a WKY standard strain, at 6 weeks old. In a preliminary study, HA-WKY but not WKY/Izm showed hyperinsulinemia and severe hypercholesterolemia when fed a high-fat diet. This study analyzed the differences between HA-WKY and WKY/Izm to investigate the causes of hyperadiponectinemia.Main methodsSix-week-old male HA-WKY and WKY/Izm were used for an analysis of adiponectin-related factors.Key findingsThe main intermediates in the adiponectin signaling pathway, AMP-activated protein kinase and peroxisome proliferator-activated receptor α, were activated at similar levels in liver and skeletal muscle between HA-WKY and WKY/Izm, although HA-WKY had not only higher adiponectin concentrations but also extremely high levels of high-molecular weight (HMW, polymer) adiponectin, which is the active form. The main difference between HA-WKY and WKY/Izm was the existence of adiponectin, mainly middle-molecular weight (MMW, hexamer) and HMW adiponectin multimers, in skeletal muscle extracts from WKY/Izm but not HA-WKY. Skeletal muscle in WKY/Izm expressed much higher amounts of T-cadherin, a receptor for MMW and HMW adiponectin multimers, than that in HA-WKY. In contrast, there was no significant difference in the expression level of adiponectin receptor 2 for trimer, MMW, and HMW adiponectin multimers.SignificanceThe results suggested that the existence of adiponectin in WKY/Izm skeletal muscle was due to the binding of serum adiponectin. The difference in serum adiponectin concentrations between HA-WKY and WKY/Izm could come from the difference in adiponectin binding to skeletal muscle.     

Adsorption of a PEO–PPO–PEO triblock copolymer on metal oxide surfaces with a view to reducing protein adsorption and further biofouling

Abstract

Biomolecule adsorption is the first stage of biofouling. The aim of this work was to reduce the adsorption of proteins on stainless steel (SS) and titanium surfaces by modifying them with a poly(ethylene oxide) (PEO)–poly(propylene oxide) (PPO)–PEO triblock copolymer. Anchoring of the central PPO block of the copolymer is known to be favoured by hydrophobic interaction with the substratum. Therefore, the surfaces of metal oxides were first modified by self-assembly of octadecylphosphonic acid. PEO–PPO–PEO preadsorbed on the hydrophobized surfaces of titanium or SS was shown to prevent the adsorption of bovine serum albumin (BSA), fibrinogen and cytochrome C, as monitored by quartz crystal microbalance (QCM). Moreover, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry were used to characterize the surfaces of the SS and titanium after competitive adsorption of PEO–PPO–PEO and BSA. The results show that the adsorption of BSA is well prevented on hydrophobized surfaces, in contrast to the surfaces of native metal oxides.     

Dye-ligand poly(GMA–TAIC–DVB) affinity adsorbent for protein adsorption

Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) was prepared by a radical suspension copolymerization. Reaction of the copolymer with 2-hydroxyethyl amine was employed to obtain a hydrophilic matrix. An affinity dye, Cibacron blue 3GA, was then coupled covalently to prepare a novel macroporous affinity adsorbent. The surface and pore structure of the affinity adsorbent were examined by scanning electron micrography (SEM). SEM observations showed that the affinity adsorbent abounded in macropores. Bovine serum albumin (BSA) and lysozyme (Lys) were used as samples to examine the adsorption properties of the adsorbent. Under appropriate conditions, the affinity adsorbent had a capacity of 15.5 mg BSA/g and 22.3 mg Lys/g (wet adsorbent weight). The adsorbed proteins could be desorbed by increasing liquid phase ionic strength or by using a NaOH solution, and the adsorbent could be recycled for protein adsorption.