Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent

In this work, we investigate the influence of crosslinkers on the operational and heat stability of immobilized enzymes on a silanized silicon surface. To this end, glucose-6-phosphate dehydrogenase (G6PDH), a model multimeric enzyme, was attached through bifunctional crosslinkers able to bind covalently the ?NH₂ in the silane layer and of amine residues in the enzyme. Five bifunctional crosslinkers in the form of “X-spacer-X” were used, differing by the reactive functional groups (X = aldehyde: ?CHO, isothiocyanate: ?NCS, isocyanate: ?NCO), by the nature of the spacer chain (aromatic or aliphatic) or by the geometry (bifunctional groups positioned in meta- or para- on an aromatic ring). A thermostability enhancement has been obtained for enzymes immobilized using 1,4-phenylene diisothiocyanate (PDC) and 1,4-phenylene diisocyanate (DIC). Moreover, using the latter crosslinker, activity was the mostly preserved upon successive uses, thus giving the best operational stability achieved. Changing the geometry of the cross-linker, i.e., 1,4- as compared to 1,3-phenylene diisothiocyanate (PDC and MDC, respectively), has a crucial effect on operational and thermal stabilities. Indeed, among all used crosslinkers, the most important loss was observed for MDC (residual activity after 6 times use is ~16%). Using dialdehyde crosslinkers: glutaraldehyde (GA) and terephtalaldehyde (TE), activity was significantly less well preserved than with DIC and PDC (for GA and TE, a loss of about 50% at 30 °C against no loss for PDC and DIC).These effects can be explained by a multipoint attachment model, in which a higher number of anchoring points stabilizes the three-dimensional structure and especially the binding of the two subunits in the active dimer, at the expense of a greater rigidity which is detrimental to the absolute activity. The differences observed with the crosslinkers are mainly due to steric hindrance at the interface which seems to be greatly influenced by the structure and the reactivity of the linkers.     

Dipetalonema viteae: Extraction and immunogenicity of cuticular antigens from female worms

The surface of adult female Dipetalonema viteae filarial worms was labeled with ¹²⁵I using 1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril as an iodinating reagent. Ultrastructural autoradiographs showed specific labeling of only the outermost cuticular layers. Surface-labeled worms were homogenized and were extracted with phosphate-buffered saline followed by NaOH. These extracts contained only minor amounts of radioactive TCA-precipitable material. Proteolytic digestion of the remaining sediment either with proteinase K, or thermolysin or subtilisin solubilized about 60% of the radioactive material of which 30% was TCA precipitable. Golden hamsters were injected with proteolytic extracts of unlabeled female worms. Subtilisin and thermolysin extracts provoked antibodies against somatic structures (e.g., gut, uterus, muscles) but not against the cuticle, whereas immunization with the proteinase K extract induced antibodies exclusively against cuticular and hypodermic structures of female and male worms.     

Adsorption of viruses to charge-modified silica.

The purpose of this study was to provide a clearer understanding of virus adsorption, focusing specifically on the role of electrostatic interactions between virus particles and adsorbent surfaces. The adsorption of poliovirus 1, reovirus types 1 and 3, and coliphages MS-2 and T2 to colloidal silica synthetically modified to carry either positive or negative surface charge was evaluated. Adsorption experiments were performed by combining virus and silica in 0.1-ionic-strength buffers of pH 4.0, 6.4, and 8.5. Samples agitated for specified adsorption periods were centrifuged to pellet adsorbent particles plus adsorbed virus, and the supernatants were assayed for unadsorbed virus. All viruses adsorbed exclusively to negatively charged silica at pH values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all viruses adsorbed exclusively to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge is negative. Viruses in near-isoelectric state adsorbed to all types of silica, albeit to a lesser degree.     

Adsorption of viruses to charge-modified silica

The purpose of this study was to provide a clearer understanding of virus adsorption, focusing specifically on the role of electrostatic interactions between virus particles and adsorbent surfaces. The adsorption of poliovirus 1, reovirus types 1 and 3, and coliphages MS-2 and T2 to colloidal silica synthetically modified to carry either positive or negative surface charge was evaluated. Adsorption experiments were performed by combining virus and silica in 0.1-ionic-strength buffers of pH 4.0, 6.4, and 8.5. Samples agitated for specified adsorption periods were centrifuged to pellet adsorbent particles plus adsorbed virus, and the supernatants were assayed for unadsorbed virus. All viruses adsorbed exclusively to negatively charged silica at pH values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all viruses adsorbed exclusively to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge is negative. Viruses in near-isoelectric state adsorbed to all types of silica, albeit to a lesser degree.     

Attaching biological probes to silica optical biosensors using silane coupling agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity/hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest. These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis. While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators. WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations. The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies. Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment. To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies/antigens) through surface modification. Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface. Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.     

Anatomy of a crosslinker

Crosslinking mass spectrometry has become a core technology in structural biology and is expanding its reach towards systems biology. Its appeal lies in a rapid workflow, high sensitivity and the ability to provide data on proteins in complex systems, even in whole cells. The technology depends heavily on crosslinking reagents. The anatomy of crosslinkers can be modular, sometimes comprising combinations of functional groups. These groups are defined by concepts including: reaction selectivity to increase information density, enrichability to improve detection, cleavability to enhance the identification process and isotope-labelling for quantification. Here, we argue that both concepts and functional groups need more thorough experimental evaluation, so that we can show exactly how and where they are useful when applied to crosslinkers. Crosslinker design should be driven by data, not only concepts. We focus on two crosslinker concepts with large consequences for the technology, namely reactive group reaction kinetics and enrichment groups.     

Solid-phase synthesis of reactive peptide crosslinker by selective deprotection

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.     

Digit ratio predicts sense of direction in women

The relative length of the second-to-fourth digits (2D:4D) has been linked with prenatal androgen in humans. The 2D:4D is sexually dimorphic, with lower values in males than females, and appears to correlate with diverse measures of behavior. However, the relationship between digit ratio and cognition, and spatial cognition in particular, has produced mixed results. In the present study, we hypothesized that spatial tasks separating cue conditions that either favored female or male strategies would examine this structure-function correlation with greater precision. Previous work suggests that males are better in the use of directional cues than females. In the present study, participants learned a target location in a virtual landscape environment, in conditions that contained either all directional (i.e., distant or compass bearing) cues, or all positional (i.e., local, small objects) cues. After a short delay, participants navigated back to the target location from a novel starting location. Males had higher accuracy in initial search direction than females in environments with all directional cues. Lower digit ratio was correlated with higher accuracy of initial search direction in females in environments with all directional cues. Mental rotation scores did not correlate with digit ratio in either males or females. These results demonstrate for the first time that a sex difference in the use of directional cues, i.e., the sense of direction, is associated with more male-like digit ratio.     

The Nature of Impulsivity: Visual Exposure to Natural Environments Decreases Impulsive Decision-Making in a Delay Discounting Task

The benefits of visual exposure to natural environments for human well-being in areas of stress reduction, mood improvement, and attention restoration are well documented, but the effects of natural environments on impulsive decision-making remain unknown. Impulsive decision-making in delay discounting offers generality, predictive validity, and insight into decision-making related to unhealthy behaviors. The present experiment evaluated differences in such decision-making in humans experiencing visual exposure to one of the following conditions: natural (e.g., mountains), built (e.g., buildings), or control (e.g., triangles) using a delay discounting task that required participants to choose between immediate and delayed hypothetical monetary outcomes. Participants viewed the images before and during the delay discounting task. Participants were less impulsive in the condition providing visual exposure to natural scenes compared to built and geometric scenes. Results suggest that exposure to natural environments results in decreased impulsive decision-making relative to built environments.     

Systematics and biology of silica bodies in monocotyledons

Many plants take up soluble monosilicic acid from the soil. Some of these plants subsequently deposit it as cell inclusions of characteristic structure. This article describes the distribution and diversity of opaline silica bodies in monocotyledons in a phylogenetic framework, together with a review of techniques used for their examination, and the ecology, function and economic applications of these cell inclusions. There are several different morphological forms of silica in monocot tissues, and the number of silica bodies per cell may also vary. The most common type is the “druse-like” spherical body, of which there is normally a single body per cell, more in some cases. Other forms include the conical type and an amorphous, fragmentary type (silica sand). Silica bodies are most commonly found either in the epidermis (e.g., in grasses, commelinas and sedges) or in the sheath cells of vascular bundles (e.g., in palms, bananas and orchids). Silica-bearing cells are most commonly associated either with subepidermal sclerenchyma or bundle-sheath sclerenchyma. Silica bodies are found only in orchids and commelinids, not in other lilioid or basal monocots. In orchids, silica bodies are entirely absent from subfamilies Vanilloideae and Orchidoideae and most Epidendroideae but present in some Cypripedioideae and in the putatively basal orchid subfamily Apostasioideae. Among commelinid monocots, silica bodies are present in all palms, Dasypogonaceae and Zingiberales but present or absent in different taxa of Poales and Commelinales, with at least four separate losses of silica bodies in Poales.     

Derivatized silica spheres as immunospecific markers for high resolution labeling in electron microscopy

For high resolution labeling of influenza virus cell surface antigens on HeLa cells, an immunospecific marker is used with silica sphere cores of 13--14 nm average diameter. These markers are formed using commercially available silica sphere sols. Two other size ranges are available, 7--8 nm and 22--25 nm. The steps for chemical derivatization are described in detail. Amino and aldehyde functions are covalently introduced onto the sphere surface. Sols of these derivatized silica spheres (DSS) are physicochemically stable and therefore usable for years. Coupling of IgG to DSS followed by permeation chromatography on controlled pore glass results in size-defined immunospecific silica sphere markers (DSS-markers). Saturation labeling of cell surface antigens on HeLa cells on cover slips is obtained with the final sphere concentration of 10(14) DSS-marker/cm3 within 20 min. With usual protective conditions, the marker stability and labeling ability are preserved for months. The visibility and the fine structure of the DSS-marker on cell surfaces are shown by using transmission electron microscopy (TEM) with stereo replicas and ultrathin sections.     

Minimal In Vitro Antimicrobial Efficacy and Ocular Cell Toxicity from Silver Nanoparticles

Silver in various forms has long been recognized for antimicrobial properties, both in biomedical devices and in eyes. However, soluble drugs used on the ocular surface are rapidly cleared through tear ducts and eventually ingested, resulting in decreased efficacy of the drug on its target tissue and potential concern for systemic side effects. Silver nanoparticles were studied as a source of anti-microbial silver for possible controlled-release contact lens controlled delivery formulations. Silver ion release over a period of several weeks from nanoparticle sources of various sizes and doses was evaluated in vitro against Pseudomonas aeruginosa strain PAO1. Mammalian cell viability and cytokine expression in response to silver nanoparticle exposure is evaluated using corneal epithelial cells and eye-associated macrophages cultured in vitro in serum-free media. Minimal microcidal and cell toxic effects were observed for several silver nanoparticle suspensions and aqueous extraction times for bulk total silver concentrations commensurate with comparative silver ion (e.g., ) toxicity. This indicates that (1) silver particles themselves in these size ranges (20–60 nm diameter) are not microcidal under conditions tested, and (2) insufficient silver ion is generated from these particles at these silver ion-equivalent loadings to produce observable biological effects compared to silver ions in these in vitro assays. This is consistent with confounding literature describing both efficacy and lack of microcidal effects for silver nanoparticles, depending on milieu, surface oxide properties, and size. If dosing allows substantially increased silver particle loading in the lens to produce sufficient pathogen-toxic silver ions and/or particle-microbe direct contact, the bactericidal efficacy of silver nanoparticles in vitro could possibly limit bacterial colonization problems associated with extended-wear contact lenses.     

Characterization of vitamin K from bovine liver

Concentrated fractions of vitamin K from bovine liver were purified by thin-layer chromatography and fractions were analyzed by UV spectroscopy and mass spectrometry. The chromatographic behavior of the purified vitamins was compared with that of known compounds on thin layers of silica gel, either untreated or impregnated with silver nitrate or paraffin. The principal forms of vitamin K recovered from bovine liver were highly lipophilic. Two fractions were obtained which collectively gave identifiable mass spectra of menaquinone-10, menaquinone-11, and menaquinone-12.     

Preparation of high-capacity supports containing protein G immobilized to porous silica

This study examined the preparation of high-capacity silica supports containing immobilized protein G. A maximum content of 39 mg protein G/g silica was obtained when using 100 Å pore size silica, followed by 33 mg/g for 50 Å silica and 9.3-24 mg/g for 300-4000 Å silica. The surface coverage of protein G increased with pore size, with a maximum level of 0.037 μmol/m² being obtained for 4000 Å silica. These supports gave comparable apparent activities (i.e., 30-47% binding to rabbit immunoglobulin G [IgG]), with the highest binding capacities (71-77 mg IgG/g silica) being obtained for 50-100 Å silica.     

A device to study the effects of stretch gradients on cell behavior

Mechanical forces are key regulators of cell function with varying loads capable of modulating behaviors such as alignment, migration, phenotype modulation, and others. Historically, cell-stretching experiments have employed mechanically simple environments (e.g., uniform uniaxial or equibiaxial stretches). However, stretch distributions in vivo can be highly non-uniform, particularly in cases of disease or subsequent to interventional treatments. Herein, we present a cell-stretching device capable of subjecting cells to controllable gradients in biaxial stretch via radial deformation of circular elastomeric membranes. By including either a defect or a rigid fixation at the center of the membrane, various gradients are generated. Capabilities of the device were quantified by tracking marked positions of the membrane while applying various loads, and experimental feasibility was assessed by conducting preliminary experiments with 3T3 fibroblasts and 10T1/2 cells subjected to 24 h of cyclic stretch. Quantitative real-time PCR was used to measure changes in mRNA expression of a profile of genes representing the major smooth muscle phenotypes. Genes associated with the contractile state were both upregulated (e.g., calponin) and downregulated (e.g., α-2-actin), and genes associated with the synthetic state were likewise both upregulated (e.g., SKI-like oncogene) and downregulated (e.g., collagen III). In addition, cells aligned with an orientation perpendicular to the maximal stretch direction. We have developed an in vitro cell culture device that can produce non-uniform stretch environments similar to in vivo mechanics. Cells stretched with this device showed alignment and altered mRNA expression indicative of phenotype modulation. Understanding these processes as they relate to in vivo pathologies could enable a more accurately targeted treatment to heal or inhibit disease, either through implantable device design or pharmaceutical approaches.     

Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides

We examined the fluorescence spectral properties of Cy3- and Cy5-labeled oligonucleotides at various distances from the surface of silver island films. The distance to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA) and avidin, followed by binding of a biotinylated oligonucleotide. The maximum enhancement of fluorescence near a factor of 12 was observed for the first BSA-avidin layer, with the enhancement decreasing to 2-fold for six layers. The minimum lifetimes were observed for the first BSA-avidin layer, and were about 25-fold shorter than on quartz slides without silver, with the lifetimes being about 2-fold shorter for six BSA-avidin layers. These results suggest that maximum fluorescence enhancements occur about 90A from the silver surface, a distance readily obtained by one or two layers of proteins.     

Detecting oxygen consumption in the proximity of Saccharomyces cerevisiae cells using self-assembled fluorescent nanosensors

We describe a strategy for the preparation and self-assembly of fluorescent nanosensors onto Saccharomyces cerevisiae cell surfaces for dynamically measuring oxygen concentration in the proximity of living cells. Amine functionalized polystyrene nanobeads were impregnated with an oxygen-sensitive ruthenium(II) complex and the beads' surface was coated with polyethylenimine. The resulting nanosensors were assembled on individual S. cerevisiae cells in a controlled manner at physiological pH for continuously monitoring oxygen consumption. This approach exemplifies a general scheme for assembling fluorescent nanosensors on cells for the non-invasive, reversible, and real-time measurement of other physiologically relevant processes, such as the efflux of protons and carbon dioxide, or the influx of glucose.     

Application of multiple response optimization design to quantum dot-encoded microsphere bioconjugates hybridization assay

The optimization of DNA hybridization for genotyping assays is a complex experimental problem that depends on multiple factors such as assay formats, fluorescent probes, target sequence, experimental conditions, and data analysis. Quantum dot-doped particle bioconjugates have been previously described as fluorescent probes to identify single nucleotide polymorphisms even though this advanced fluorescent material has shown structural instability in aqueous environments. To achieve the optimization of DNA hybridization to quantum dot-doped particle bioconjugates in suspension while maximizing the stability of the probe materials, a nonsequential optimization approach was evaluated. The design of experiment with response surface methodology and multiple optimization response was used to maximize the recovery of fluorescent probe at the end of the assay simultaneously with the optimization of target–probe binding. Hybridization efficiency was evaluated by the attachment of fluorescent oligonucleotides to the fluorescent probe through continuous flow cytometry detection. Optimal conditions were predicted with the model and tested for the identification of single nucleotide polymorphisms. The design of experiment has been shown to significantly improve biochemistry and biotechnology optimization processes. Here we demonstrate the potential of this statistical approach to facilitate the optimization of experimental protocol that involves material science and molecular biology.     

Closely related species of birds differ more in body size when their ranges overlap—in warm,but not cool,climates

Differences in body size are widely thought to allow closely related species to coexist in sympatry, but body size also varies as an adaptive response to climate. Here, we use a sister lineage approach to test the prediction that body size differences between closely related species of birds worldwide are greater for species whose ranges are sympatric rather than allopatric. We further test if body size differences among sympatric versus allopatric species vary with geography, evolutionary distance, and environmental temperatures. We find greater differences in size among sympatric compared with allopatric lineages, but only in closely related species that live where mean annual temperatures are above 25°C. These size differences in warm environments declined with the evolutionary distance between sister lineages. In species living in cooler regions, closely related allopatric and sympatric species did not differ significantly in size, suggesting either that colder temperatures constrain the evolutionary divergence of size in sympatry, or that the biotic selective pressures favoring size differences in sympatry are weaker in colder environments. Our results are consistent with suggestions by Wallace, Darwin, and Dobzhansky that climatic selective pressures are more important in cooler environments (e.g., high elevations and latitudes) whereas biotic selective pressures dominate in warm environments (e.g., lowland tropics).