In the present study, the productions of antinociception induced by acute and chronic immobilization stress were compared in several animal pain models. In the acute immobilization stress model (up to 1 hr immobilization), the antinociception was produced in writhing, tail-flick, and formalin- induced pain models. In chronic immobilization stress experiment, the mouse was enforced into immobilization for 1 hr/day for 3, 7, or 14 days, then analgesic tests were performed. The antinociceptive effect was gradually reduced after 3, 7 and 14 days of immobilization stress. To delineate the molecular mechanism involved in the antinociceptive tolerance development in the chronic stress model, the expressions of some signal molecules in dorsal root ganglia (DRG), spinal cord, hippocampus, and the hypothalamus were observed in acute and chronic immobilization models. The COX-2 in DRG, p-JNK, p-AMPKα1, and p-mTOR in the spinal cord, p-P38 in the hippocampus, and p-AMPKα1 in the hypothalamus were elevated in acute immobilization stress, but were reduced gradually after 3, 7 and 14 days of immobilization stress. Our results suggest that the chronic immobilization stress causes development of tolerance to the antinociception induced by acute immobilization stress. In addition, the COX-2 in DRG, p-JNK, p-AMPKα1, and p-mTOR in the spinal cord, p-P38 in the hippocampus, and p-AMPKα1 in the hypothalamus may play important roles in the regulation of antinociception induced by acute immobilization stress and the tolerance development induced by chronic immobilization stress.

Nowadays, commercial erythritol synthesis is performed by free-cell fermentation with fungi in liquid media containing high concentrations of pure carbon sources. Alternative fermentation techniques, such as cell immobilization, could imply an economic and energetic improvement for erythritol-producing factories. The present work describes, for the first time, the feasibility of achieving cell immobilization during erythritol production. Cells of the fungus Moniliella pollinis were successfully immobilized on a cotton cloth which was placed inside a 2-L bioreactor, where they were fed with red grape must supplemented with yeast extract. They produced 47.03 ± 6.16 g/L erythritol in 96 h (yield 0.18 ± 0.04 g/g) over four consecutive fermentation batches. The immobilized cells remained stable and operative during a 456 h period. The erythritol concentration attained was similar (p > 0.05; Tukey HSD test) to the reference value obtained with the use of free cells (41.88 ± 5.18 g/L erythritol) under the same fermentation conditions. The comparable results observed for free and immobilized cells evidences the efficiency of the immobilization system. Therefore, the proposed method for erythritol bioproduction eliminates the need for the continuous preparation of fungal inocula before each fermentation batch, thus reducing the costs of the reagents and energy.

Enzyme immobilization is a widespread empiric technology to achieve more stable, active and reusable enzymes. The empiricism can be reduced by the application of rational design procedures employing bioinformatic tools, engineered-proteins and detailed analyses of existent data. In this chapter, we describe relevant approaches to rationalize the design of enzyme immobilization protocols, with special attention to the modulation of immobilization pH to regulate the operational stability of glutaraldehyde cross-linked enzymes and the coating of iron-containing supports to preserve the integrity of iron-sensitive enzymes. Other strategies, such as the use of factorial planning, optimization of specific enzyme orientation through protein engineering and the use of mathematical algorithms and in silico prediction tools are also described to reduce the classical empiricism. Finally, a public repository creation is proposed as a new promising tool to develop an improvement on future rational design procedures of enzyme immobilization.

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit[7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

Different amounts of sodium-alendronate (ALN) were loaded into layered zirconium phosphates of alpha and gamma type (αZP and γZP) by means of topotactic exchange reactions of phosphate with ALN. In order to extend the exchange process to the less accessible interlayer regions, ALN solutions were contacted with colloidal dispersions of the layered solids previously exfoliated in single sheets by means of intercalation reaction of propylamine (for αZP) or acetone (for γZP). The ALN loading degree was determined by liquid P-nuclear magnetic resonance (NMR) and inductively coupled plasma (ICP), and it was reported as ALN/Zr molar ratios (Rs). The maximum R obtained for γZP was 0.34, while αZP was able to load a higher amount of ALN, reaching Rs equal to 1. The synthesized compounds were characterized by X-ray powder diffractometry, scanning electron microscopy (SEM), solid-state NMR, and infrared spectroscopy. The way the grafted organo-phosphonate groups were bonded to the layers of the host structure was suggested. The effect of ZP derivatives was assessed on cell proliferation, and the results showed that after 7 days of incubation, none of the samples showed a decrease in cell proliferation.

Motor imagery (MI) is known to engage motor networks and is increasingly used as a relevant strategy in functional rehabilitation following immobilization, whereas its effects when applied during immobilization remain underexplored. Here, we hypothesized that MI practice during 11 h of arm-immobilization prevents immobilization-related changes at the sensorimotor and cortical representations of hand, as well as on sleep features. Fourteen participants were tested after a normal day (without immobilization), followed by two 11-h periods of immobilization, either with concomitant MI treatment or control tasks, one week apart. At the end of each condition, participants were tested on a hand laterality judgment task, then underwent transcranial magnetic stimulation to measure cortical excitability of the primary motor cortices (M1), followed by a night of sleep during which polysomnography data was recorded. We show that MI treatment applied during arm immobilization had beneficial effects on (1) the sensorimotor representation of hands, (2) the cortical excitability over M1 contralateral to arm-immobilization, and (3) sleep spindles over both M1s during the post-immobilization night. Furthermore, (4) the time spent in REM sleep was significantly longer, following the MI treatment. Altogether, these results support that implementing MI during immobilization may limit deleterious effects of limb disuse, at several levels of sensorimotor functioning.

Non-thermal plasmas are used in various applications to inactivate biological agents or biomolecules. A complex cocktail of reactive species, (vacuum) UV radiation and in some cases exposure to an electric field together cause the detrimental effects. In contrast to this disruptive property of technical plasmas, we have shown previously that it is possible to use non-thermal plasma-generated species such as H2O2 as cosubstrates in biocatalytic reactions. One of the main limitations in plasma-driven biocatalysis is the relatively short enzyme lifetime under plasma-operating conditions. This challenge could be overcome by immobilizing the enzymes on inert carrier materials. Here, we tested whether immobilization is suited to protect proteins from inactivation by plasma. To this end, using a dielectric barrier discharge device (PlasmaDerm), plasma stability was tested for five enzymes immobilized on ten different carrier materials. A comparative analysis of the treatment times needed to reduce enzyme activity of immobilized and free enzyme by 30% showed a maximum increase by a factor of 44. Covalent immobilization on a partly hydrophobic carrier surface proved most effective. We conclude from the study, that immobilization universally protects enzymes under plasma-operating conditions, paving the way for new emerging applications.

Antibodies have been shown to hinder the movement of herpes simplex virus virions in cervicovaginal mucus, as well as other viruses in other mucus secretions. However, it has not been possible to directly observe the mechanisms underlying this phenomenon, so the nature of virion-antibody-mucin interactions remain poorly understood. In this work, we analyzed thousands of virion traces from single particle tracking experiments to explicate how antibodies must cooperate to immobilize virions for relatively long time periods. First, using a clustering analysis, we observed a clear separation between two classes of virion behavior: freely diffusing and immobilized. While the proportion of freely diffusing virions decreased with antibody concentration, the magnitude of their diffusivity did not, implying an all-or-nothing dichotomy in the pathwise effect of the antibodies. Proceeding under the assumption that all binding events are reversible, we used a novel switch-point detection method to conclude that there are very few, if any, state switches on the experimental timescale of 20 s. To understand this slow state switching, we analyzed a recently proposed continuous-time Markov chain model for binding kinetics and virion movement. Model analysis implied that virion immobilization requires cooperation by multiple antibodies that are simultaneously bound to the virion and mucin matrix and that there is an entanglement phenomenon that accelerates antibody-mucin binding when a virion is immobilized. In addition to developing a widely applicable framework for analyzing multistate particle behavior, this work substantially enhances our mechanistic understanding of how antibodies can reinforce a mucus barrier against passive invasive species.

Laccase from Pycnoporus sanguineus CS43 was successfully immobilized onto Immobead-150 and Eupergit-C by covalent binding and by entrapment in LentiKats. The highest immobilization was onto Immobead-150 (97.1±1.2%) compared to the other supports, LentiKats (89±1.1%) and Eupergit-C (83.2±1.4%). All three immobilized enzyme systems showed increased thermostability and better mechanical properties than free laccase. Moreover, after 5 cycles of reuse of these systems, 90% of initial laccase activity was retained. Immobead-150 and LentiKats systems exhibited the highest efficiencies in removal of m-cresol under the combined actions of biodegradation and adsorption, while laccase entrapped in LentiKats showed a high ability for degradation of m-cresol within 24h. In addition, the typical Michaelis-Menten enzymatic model effectively described the kinetic profile of m-cresol degradation by the enzyme entrapped in LentiKats. Based on the results obtained in the present study, it can be established that the immobilized biocatalysts developed here possess significant potential for wastewater treatment.

Metronidazole (MTZ) is a widely used drug, but due to its many side effects, there is a growing trend today to use a minimum dose while maintaining high efficacy. One way to meet this demand is to reduce the size of the drug particles. A relatively new method of size reduction is attaching the drug molecules to a mesoporous carrier. In this paper, we studied the fixation of MTZ molecules on mesoporous silica carriers. The drug was immobilized on two mesoporous silica materials (Syloid, SBA-15) with the use of a variety of immersion techniques and solvents. The immobilized drug was subjected to physicochemical examinations (e.g., SEM, XPS, XRD, nitrogen uptake, DSC) and dissolution studies. A significantly higher immobilization was attained on SBA-15 than on a Syloid carrier. Among the processing parameters, the type of MTZ solvent had the highest influence on immobilization. Ultrasonic agitation had a lower but still significant impact, while the concentration of MTZ in the solution made no difference. Under optimal conditions, with the application of an ethyl acetate solution, the surface coverage on SBA-15 reached as much as 91%. The immobilized MTZ exhibited a ca. 10% faster dissolution rate as compared to the pure micron-sized drug particles.

The purpose of this study was to evaluate and quantify the interfraction reproducibility and intrafraction immobilization precision of a modified GTC frame. The error of the patient alignment and imaging systems were measured using a cranial skull phantom, with simulated, predetermined shifts. The kV setup images were acquired with a room-mounted set of kV sources and panels. Calculated translations and rotations provided by the computer alignment software relying upon three implanted fiducials were compared to the known shifts, and the accuracy of the imaging and positioning systems was calculated. Orthogonal kV setup images for 45 proton SRT patients and 1002 fractions (average 22.3 fractions/patient) were analyzed for interfraction and intrafraction immobilization precision using a modified GTC frame. The modified frame employs a radiotransparent carbon cup and molded pillow to allow for more treatment angles from posterior directions for cranial lesions. Patients and the phantom were aligned with three 1.5 mm stainless steel fiducials implanted into the skull. The accuracy and variance of the patient positioning and imaging systems were measured to be 0.10 ± 0.06 mm, with the maximum uncertainty of rotation being ±0.07°. 957 pairs of interfraction image sets and 974 intrafraction image sets were analyzed. 3D translations and rotations were recorded. The 3D vector interfraction setup reproducibility was 0.13 mm ± 1.8 mm for translations and the largest uncertainty of ± 1.07º for rotations. The intrafraction immobilization efficacy was 0.19 mm ± 0.66 mm for translations and the largest uncertainty of ± 0.50º for rotations. The modified GTC frame provides reproducible setup and effective intrafraction immobilization, while allowing for the complete range of entrance angles from the posterior direction.

(1) Submitochondrial particles prepared from beef liver mitochondria were immobilized on Fractosil, a porous form of silica, in order to stabilize their enzymatic activity. (2) The catalytic activity of succinate-cytochrome c reductase, an enzyme complex of the inner mitochondrial membrane, was followed in this study. Adsorption resulted in significant stabilization with a lowering of K(m) (app.) for succinate, in spite of mass transfer and diffusion limitations expected to occur in such a complex and heterogeneous system. An increase in catalytic potential was also observed upon immobilization. These observations, taken together, suggest that substantial degree of conversation of substrates to their respective products may be achieved by such immobilized preparations. (3) Positive cooperative interactions for binding of submitochondrial particles to the matrix was observed, apparently with two sets of sites, the second set indicating a much greater hill coefficient. (4) The present report indicates that adsorption with the use of a porous inorganic support such as Fractosil may provide a simple and efficient method of immobilization. Such preparations containing membrane enzymes in their native microenvironments would be useful for continuous catalytic transformations and also for construction of biosensors.

Muscle atrophy due to fragility fractures or frailty worsens not only activity of daily living and healthy life expectancy, but decreases life expectancy. Although several therapeutic agents for muscle atrophy have been investigated, none is yet in clinical use. Here we report that bezafibrate, a drug used to treat hyperlipidemia, can reduce immobilization-induced muscle atrophy in mice. Specifically, we used a drug repositioning approach to screen 144 drugs already utilized clinically for their ability to inhibit serum starvation-induced elevation of Atrogin-1, a factor related to muscle atrophy, in myotubes in vitro. Two candidates were selected, and here we demonstrate that one of them, bezafibrate, significantly reduced muscle atrophy in an in vivo model of muscle atrophy induced by leg immobilization. In gastrocnemius muscle, immobilization reduced muscle weight by an average of ~ 17.2%, and bezafibrate treatment prevented ~ 40.5% of that atrophy. In vitro, bezafibrate significantly inhibited expression of the inflammatory cytokine Tnfa in lipopolysaccharide-stimulated RAW264.7 cells, a murine macrophage line. Finally, we show that expression of Tnfa and IL-1b is induced in gastrocnemius muscle in the leg immobilization model, an activity significantly antagonized by bezafibrate administration in vivo. We conclude that bezafibrate could serve as a therapeutic agent for immobilization-induced muscle atrophy.

Enforced limb disuse strongly disrupts the cortical networks that are involved in sensorimotor activities. This disruption causes a cortical reorganization that may be functionally maladaptive. In this study, we used functional magnetic resonance imaging (fMRI) to investigate whether it is possible to prevent this reorganization by compensating for the lack of actual kinesthetic perception with illusory movements induced by "neuromimetic" proprio-tactile feedback that is artificially delivered during immobilization. Sixteen healthy volunteers were equipped for five days with full-hand ortheses that prevented them from performing finger and hand movements but allowed for kinesthetic and tactile sensations. Eight participants received a twice-daily proprio-tactile treatment consisting of the perception of kinesthetic sensations resembling those felt during actual movements generated by miniature vibrators set in the ortheses at the finger and wrist levels. Eight untreated participants received no stimulation. The effects of hand immobilization and treatment were assessed by fMRI during a calibrated voluntary hand movement task and hand tactile stimulation before cast placement and immediately after cast removal. We found that the sensorimotor network was preserved in subjects who underwent this treatment during hand immobilization, while the sensorimotor network of untreated subjects was significantly altered. These findings suggest that sensory feedback and associated movement perception may counteract disuse-induced cortical plastic changes through recruitment of a large part of the cortical network used for actual performed movement. The possibility of guiding cortical plasticity with proprioceptive augmented feedback is potentially relevant for rehabilitation efforts.

Ferritin possess favorable properties because its exterior and interior surface can be applied to generate functional nanomaterials, which make them possible for enzyme immobilization and recycling. Here, we report the noncovalent immobilization of a genetically modified β-glucosidase onto the outer surface of synthetic magnetoferritin through the electrostatic interaction of a heterodimeric coiled-coil protein formed by coils containing lysine residues (K-coils) and coils containing glutamic acid (E-coils). The immobilized enzyme was characterized, and its enzymatic properties were evaluated. Furthermore, reusability of immobilized enzyme was demonstrated in aqueous solution under an applied magnetic field. The results showed that magnetoferritin was successfully prepared and it was an excellent support for enzyme immobilization. After three times usages, the retention rates were 93.75%, 82.5%, and 56.25%, respectively, demonstrating that immobilized enzyme possessed good retention efficiency and could be used as potential carrier for other biomolecules. The strategy of enzyme immobilization developed in this work can be applied, in general, to many other target molecules.

Magnetite Fe3O4 nanoparticles (NPs) were prepared by chemical coprecipitation method. Silica-coated magnetite NPs were prepared by sol-gel reaction, subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction, and then were activated with 2,4,6-trichloro-1,3,5-triazine (TCT) and covalently immobilized with bovine serum albumin (BSA). The size and structure of the particles were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and dynamic light scattering (DLS) techniques. The immobilization was confirmed by Fourier transform infrared spectroscopy (FT-IR). XRD analysis showed that the binding process has not done any phase change to Fe3O4. The immobilization time for this process was 4 h and the amount of immobilized BSA for the initial value of 1.05 mg BSA was about 120 mg/gr nanoparticles. Also, the influences of three different buffer solutions and ionic strength on covalent immobilization were evaluated.

[Purpose] The purpose of our study was to clarify temporal effects on restrictions to range of motion and the histopathological changes of joint components after joint immobilization in a rat knee-contracture model. [Subjects] Fifty-four male Wistar rats were randomly divided into two groups: a fixation group, and a control group. [Methods] In the fixation group, unilateral knee joints were immobilized at full flexion using a plaster cast for 4 weeks. At four weeks the animals were randomly divided into six subgroups, corresponding to the time of examination after cast removal: 0, 4, 8, 16, 24, and 32 weeks. For comparison, control group animals of corresponding age were also examined. [Results] Although movement restrictions of the knee joint had completely recovered 6 weeks after the cast removal, cartilage and synovial membrane structures did not completely recover. [Conclusion] These findings have not previously been reported, and as they form an addition to the fundamental scientific foundations of physical therapy, further research must examine these findings from a variety of perspectives.

We recently reported that the water holding capacity of myofibrillar protein hydrogels could be increased upon addition of small amounts of microparticles, particularly glass microspheres. Glass microspheres were found to decrease the spin-spin relaxation time (T2) of water protons in the gels, which was interpreted as enhanced water binding by the glass. We were thus interested in determining whether the observed effects on water proton relaxation were a direct consequence of water-glass interactions. Here we show how glass microspheres reduce the mobility of pure water, reflected in large decreases in the T2 of water protons, decreases in the self-diffusion coefficient of water molecules, a lower water activity, and strengthening of O-H bonds. Even though glass is considered an inert material, glass microspheres were shown to inhibit the growth of human embryonic kidney cells, and stimulate or inhibit the growth of leukemia and monocytic lymphoma cells in vitro, depending on dose and time. The germination of alfalfa seeds and the growth of E.coli cells were also inhibited upon exposure to glass microspheres. This work indicates that the properties and behavior of materials, even ones considered inert, can be affected by their size. These observations suggest possible toxicological consequences of exposure to microparticles, but also open us possibilities to affect cellular/organism function via modulation of macromolecular hydration.

Monolayers of colloidal particles trapped at an interface between two immiscible fluids play a pivotal role in many applications and act as essential models in fundamental studies. One of the main advantages of these systems is that non-close packed monolayers with tunable inter-particle spacing can be formed, as required, for instance, in surface patterning and sensing applications. At the same time, the immobilization of particles locked into desired structures to be transferred to solid substrates remains challenging. Here, we describe three different strategies to immobilize monolayers of polystyrene microparticles at water⁻decane interfaces. The first route is based on the leaking of polystyrene oligomers from the particles themselves, which leads to the formation of a rigid interfacial film. The other two rely on in situ interfacial polymerization routes that embed the particles into a polymer membrane. By tracking the motion of the colloids at the interface, we can follow in real-time the formation of the polymer membranes and we interestingly find that the onset of the polymerization reaction is accompanied by an increase in particle mobility determined by Marangoni flows at the interface. These results pave the way for future developments in the realization of thin tailored composite polymer-particle membranes.

Micrometer resolution placement and immobilization of probe molecules is an important step in the preparation of biochips and a wide range of lab-on-chip systems. Most known methods for such a deposition of several different substances are costly and only suitable for a limited number of probes. In this article we present a flexible procedure for simultaneous spatially controlled immobilization of functional biomolecules by molecular ink lithography.