Protein Released Research Articles

Encapsulated Mn-Saturated Lactoferrin as a Safe Source of Manganese Ions for Restoring Probiotic Lactobacillus plantarum.

The growth of Lactobacillus plantarum, a member of the Lactobacillus genus, which plays a crucial role in the bacterial microbiome of the gut, is significantly influenced by manganese ions. They can be safely delivered to the intestines by exploiting the chelating abilities of lactoferrin. The aim of this work was to encapsulate lactoferrin saturated with manganese ions (MnLf) in a system based on the Eudragit® RS polymer to protect protein from degradation and manganese release in the gastric environment. The entrapment efficiency was satisfactory, reaching about 95%, and most importantly, manganese ions were not released during microparticles (MPs) formation. The release profile of the protein from the freshly prepared MPs was sustained, with less than 15% of the protein released within the first hour. To achieve similar protein release efficiency, freeze-drying was carried out in the presence of 10% (w/v) mannitol as a cryoprotectant for MPs frozen at -20 °C. MPs with encapsulated MnLf exhibited prebiotic activity towards Lactobacillus plantarum. More importantly, the presence of equivalent levels of manganese ions in free form in the medium, as well as chelating by lactoferrin encapsulated in MPs, had a similar impact on stimulating bacterial growth. This indicates that the bioavailability of manganese ions in our prepared system is very good.

Antimicrobial Potency and E. coli β-Carbonic Anhydrase Inhibition Efficacy of Phenazone-Based Molecules.

In this investigation, 4-antipyrinecarboxaldhyde was reacted with methyl hydrazinecarbodithioate to afford the carbodithioate derivative 3. The as-prepared carbodithioate derivative 3 is considered to be a key molecule for the preparation of new antipyrine-1,3,4-thiadiazole-based molecules (4-9) through its reaction with the appropriate hydrazonoyl halides. Furthermore, a typical Biginelli three-component cyclocondensation reaction involving ethyl acetoacetate, 4-antipyrinecarboxaldhyde, and thiourea under the standard conditions is carried out in the presence of sulfuric acid to afford the corresponding antipyrine-pyrimidine hybrid molecule (10). The latter was submitted to react with hydrazine monohydrate to provide the corresponding hydrazide derivative (11) which, under reaction with ethyl acetoacetate in refluxing ethanol containing catalytic amount of acetic acid, afforded the corresponding derivative (12). The structure of the newly synthesized compounds was affirmed by their spectral and microanalytical data. We also screened for their antimicrobial potential (ZOI and MIC) and conducted a kinetic study. Additionally, the mechanism of biological action was assessed by a membrane leakage assay and SEM imaging technique. Moreover, the biological activities and the binding modes of these compounds were further supplemented by an in silico docking study against E. coli β-carbonic anhydrase. The amount of cellular protein released by E. coli is directly correlated to the concentration of compound 9, which was found to be 177.99 µg/mL following treatment with 1.0 mg/mL of compound 9. This finding supports compound 9's antibacterial properties and explains how the formation of holes in the E. coli cell membrane results in the release of proteins from the cytoplasm. The newly synthesized compounds represent acceptable antimicrobial activities with potential action against E. coli β-carbonic anhydrase. The docking studies and antimicrobial activity test proved that compound (9) declared a greater activity than the other synthesized compounds.

Extraction of soluble proteins by bead milling from Tetraselmis chui in two different physiological states

Tetraselmis is an interesting microalga for food products because of its high protein content and surface productivity as well as the possibility to scale up the culture easily. However, this specie is yet understudied compared to other algae such as Chlorella. Different batches of Tetraselmis chui were received from the same supplier, with different morphological properties, probably associated with a difference in physiological state: one batch contained circular and dividing cells and the other flagellated cells. The batches were characterized biochemically in terms of protein, carbohydrate and ash content and the rupturability was tested to characterize their behavior to cell disruption. One batch was found way more fragile than the other. The bead milling of the two batches showed also striking differences in terms of cell disruption kinetic, as well as dry matter repartition after centrifugation and protein recovery yields within the supernatant. Nearly no differences in protein yield were observed after bead milling in comparison to freeze-thawing for the resistant batch, with a final yield of 11 % of the initial proteins. However, a clear protein release was observed for the fragile batch along the milling residence time, with a final protein yield of 32 %. The protein released were characterized by SDS PAGE and zymography and differences were observed, confirming the difference in metabolism. Several physicochemical modifications were applied to the cell lysate to increase the yields using pH, temperature and dilution. This latest modification was the most promising. The results underlined that a difference in physiological state of Tetraselmis chui had a significant impact on the following biorefinery, affecting directly the cell disruption and the protein recovery yields. The valorization of the whole microalgae thus necessitates the finest coupling between upstream and downstream.

Meteorin-like levels are associated with active brown adipose tissue in early infancy.

Meteorin-like (METRNL) is a hormonal factor released by several tissues, including thermogenically active brown and beige adipose tissues. It exerts multiple beneficial effects on metabolic and cardiovascular systems in experimental models. However, the potential role of METRNL as brown adipokine in humans has not been investigated previously, particularly in relation to the metabolic adaptations taking place in early life, when brown adipose tissue (BAT) is particularly abundant. METRNL levels, as well as body composition (DXA) and circulating endocrine-metabolic variables, were assessed longitudinally in a cohort of infants at birth, and at ages 4 and 12 months. BAT activity was measured by infrared thermography at age 12 months. METRNL levels were also determined cross-sectionally in adults; METRNL gene expression (qRT-PCR) was assessed in BAT and liver samples from neonates, and in adipose tissue and liver samples form adults. Simpson-Golabi-Behmel Syndrome (SGBS) adipose cells were thermogenically activated using cAMP, and METRNL gene expression and METRNL protein released were analysed. Serum METRNL levels were high at birth and declined across the first year of life albeit remaining higher than in adulthood. At age 4 and 12 months, METRNL levels correlated positively with circulating C-X-C motif chemokine ligand 14 (CXCL14), a chemokine released by thermogenically active BAT, but not with parameters of adiposity or metabolic status. METRNL levels also correlated positively with infrared thermography-estimated posterior-cervical BAT activity in girls aged 12 months. Gene expression analysis indicated high levels of METRNL mRNA in neonatal BAT. Thermogenic stimulus of brown/beige adipocytes led to a significant increase of METRNL gene expression and METRN protein release to the cell culture medium. Circulating METRNL levels are high in the first year of life and correlate with indices of BAT activity and with levels of an established brown adipokine such as CXCL14. These data, in addition with the high expression of METRNL in neonatal BAT and in thermogenically-stimulated brown/beige adipocytes, suggest that METRNL is actively secreted by BAT and may be a circulating biomarker of BAT activity in early life.

Effect of Over‐the‐Counter Tooth Whitening Consumer Products of Release of Collagen and Protein from Teeth

While tooth whitening used to consist of either whitening strips or professional treatments: there are now a variety of products available to consumers in the United States. While most involve either hydrogen peroxide or its precursor, carbamide peroxide, as the active ingredients, there are alternatives. Carbamide peroxide can be administered in gel form in a tray with or without an LED light. Instead of a strip, hydrogen peroxide can now by applied directly to the teeth in form of pen and remains overnight. The goal of this project was to examine the effect of whitening agents on the release of both collagen and other proteins. Extracted molars from patients who signed informed consent forms were placed in artificial saliva and treated with activated charcoal, carbamide peroxide with (35%) or without (44%) an LED light, or a whitening pen with 14% hydrogen peroxide according to manufacturer’s instructions. Artificial saliva was changed daily, and all teeth received six days of no treatment and four rounds of 6 treatments with a break between the second and third. The fluid surrounding the teeth underwent a trichloroacetic acid precipitation to remove the non‐ collagen protein. The supernatant was adjusted to the proper pH and collagen was measured using a modified version of the Lowry assay. In addition, the proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Molars that received no treatment, as well as prior to treatment, show no protein. All treated teeth showed collagen and non‐collagen protein released whenever treatments were applied as well as in between rounds of treatment. The amount of collagen released from activated charcoal, 35% carbamide peroxide with LED, 44% carbamide peroxide and 14% hydrogen peroxide pen in mg/ml were 1.048, 1.994, 1.530, and 0.9034 respectively. For non‐collagen, the average amount of protein released for activated charcoal, 35% carbamide peroxide with LED, and 44% carbamide peroxide in mg/ml respectively were: 0.5714, 0.4155, and 0.6363. Both collagen and non‐collagen were released from all treated teeth in between rounds of treatments. The size of the proteins released was characterized using SDS PAGE. Consistent with the TCA and modified Lowry results, no bands were observed in artificial saliva prior to treatment but all treatments resulted in bands at molecular weight (MW) of 129 and 75 kDa among others. The MW of former band matches the expected subunit MW of collagen which is found in the dentin of teeth. These results demonstrate that treatment of teeth with hydrogen peroxide, carbamide peroxide with or without LED light or activated charcoal results in release of various proteins from dentin and enamel. In addition, these proteins continued to be released after treatments were applied. Hydrogen peroxide was measured using a colorimetric test. The amounts of peroxide in other samples was consistent with manufacturer’s claims.

Roles of high mobility group box 1 protein released from endothelial cells with hypoxic injury on neuronal amyloidogenesis

Hypoxia-reperfusion injury is one of the major risk factors for neurodegeneration. However, it is unclear whether ischaemic damage in brain microvascular endothelial cells plays roles in neurodegeneration, particularly in the amyloidogenic changes contributing to the development of Alzheimer's disease (AD) pathologies. Therefore, we investigated the roles of hypoxia-reoxygenation (H/R)-induced release of high mobility group box protein 1 (HMGB1), a risk molecule for AD pathogenesis in the ischaemic damaged brain, from human brain microvascular endothelial cells (HBMVECs) in neuronal amyloid beta (Aβ) production. H/R experiments were carried out in a purpose-built hypoxia glove-box chamber with continuous monitoring of O2 concentration. HBMVECs were treated with hypoxia (0.5% O2 ) and glucose deprivation for 1 h, and then the cells were restored to 37 °C with fresh M199 medium in a humidified atmosphere of 95% air and 5% CO2 . Endothelial permeability was assessed by measuring FITC-tagged dextran flux across monolayers of cultured HBMVECs. H4swe or SH-SY5Y cells were exposed to CM from HBMVECs with or without H/R for 24 h. Aβ40 and Aβ42 levels in the CM of H4swe cells were determined with commercial ELISA kits. Transfections of HMGB1 or Sirt1 siRNAs were performed using RNAi Max. Plasmids encoding Flag-tagged WT or dominant negative (DN) mutant H363Y SIRT1 were transfected using Lipofectamine. H/R increased nuclear-cytosolic translocation and secretion of HMGB1 in HBMVECs, along with increased permeability and HMGB1-dependent p-c-Jun activation. In addition, H/R increased the expression of Sirtuin 1 (Sirt1), coincident with an increase of intracellular Sirt1-HMGB1 binding in HBMVECs. H/R increased the acetylation of HMGB1 and extracellular secretion, which was significantly inhibited by Sirt1 overexpression. Furthermore, Sirt1 contributed to autophagy-mediated endogenous HMGB1 degradation. More importantly, treatment of neuronal cells with conditioned medium from H/R-stimulated HBMVECs (H/R-CM) activated their amyloidogenic pathways. The neuronal amyloidogenic changes (i.e., increased levels of extracellular Aβ40 and Aβ42) by H/R-CM from HBMVECs were further increased by Sirt1 inhibition, which was significantly suppressed by neutralization of the HMGB1 in H/R-CM. Our results suggest that HMGB1 derived from H/R-stimulated HBMVECs contributes to amyloidogenic pathways in neurons playing roles in the pathogenesis of AD, which are regulated by endothelial Sirt1.

Mitochondrial Permeability Transition Induces Skeletal Muscle Atrophy in Single Living Myofibers

Introduction In advanced age (≥75 y), skeletal muscle atrophy leads to physical frailty, impaired mobility, and increased mortality. Mitochondrial dysfunction has been linked to aging skeletal muscle atrophy, although the specific mechanisms are unclear. Mitochondrial permeability transition (MPT) has been implicated in aging biology, which involves formation of a pore across the inner mitochondrial membrane that causes dissipation of mitochondrial membrane potential and cytoplasmic release of reactive oxygen species (ROS) and mitochondrial proteins. Although we find mitochondria are sensitized to MPT in aging skeletal muscle, the role of MPT in aging muscle biology has not been explored. To gain insight to the impact of MPT in aging muscle, we recently examined the cross-sectional area (CSA) of muscle fibers with myonuclei that contained apoptosis inducing factor (AIF), a protein released from mitochondria during MPT. On average, fibers containing AIF-positive myonuclei had smaller CSA, suggesting a link between MPT and muscle atrophy in advanced age. To more directly test the involvement of MPT in muscle atrophy, we used cultured mouse skeletal muscle fibers to determine if acute chemical induction of MPT would cause fiber atrophy that would be prevented by MPT inhibition. We also hypothesized that treatment with an MPT inhibitor would prevent atrophy in a model of muscle fiber ‘disuse’ where isolated single mouse myofibers were maintained in culture conditions for 5 d. Methods Single fibers were isolated from mouse flexor digitorum brevis (FDB) muscles, plated, and treated with DMSO (vehicle control), doxorubicin (DOX; 1µM), a chemotherapy agent known to induce MPT, or DOX plus the MPT inhibitor cyclosporine A (CsA; 1 µM). Fibers were imaged at 20x upon plating and after 24h treatment. Myofiber diameter was assessed in ImageJ (average of 10 diameter measurements per fiber). Individual fibers were matched pre- to post-treatment for determination of percent change in myofiber diameter. Previous studies have established that maintaining single isolated muscle fibers in culture conditions is associated with fiber atrophy and can therefore be used as a model of disuse/denervation atrophy. On this basis, we isolated single mouse FDB myofibers and treated them with vehicle (DMSO) or 1 µM CsA every 24h under standard culture conditions for 5 d. Myofiber diameter was determined at baseline and day 5, as described above. Results Whereas 24h treatment of myofibers with DOX induced significant atrophy compared to baseline (percent change in myofiber diameter; DOX: -16.64 ± 7.78%, P<0.05), the addition of CsA prevented atrophy over this brief period (DOX+CsA: 0.22 ± 15.38%, NS). Similarly, in a model of muscle disuse, MPT inhibition using CsA prevented myofiber atrophy (baseline: 38.11 ± 5.87 µm vs. day 5: 36.49 ± 7.66 µm, NS) compared with DMSO (baseline: 35.65 ± 7.30 µm vs. day 5: 31.15 ± 7.01 µm, P<0.05). Discussion Consistent with our hypotheses, both inducing MPT using DOX and 5 days of muscle disuse led to atrophy in single living myofibers that was prevented by MPT inhibition. Therefore, our results identify MPT as a novel mechanism of skeletal muscle atrophy and a mechanism that appears relevant to causing the atrophy seen in aging skeletal muscle.

Effects of disease and dexamethasone on human cartilage breakdown kinetics

Effects of disease and dexamethasone on human cartilage breakdown kinetics

The influence of a swab type on the results of point-of-care tests

Most point-of-care tests (POCT) use swabs for sampling and/or for applying a sample on the test. A variety of swabs differing in tip materials is commercially available. Different tip materials have different chemical and physical characteristics which might influence the specimen collection and release. We investigated properties of various types of swabs used in clinical diagnostics with focusing on two kinds of analytes, DNA and proteins, which are most often used targets in POCT. As the model samples we used diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and bacterial strains of Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 for investigating the recovery of nucleic acids. We investigated four types of swabs most commonly used in clinical diagnostics in terms of absorption capacity and efficiency of release of nucleic acids and proteins. Volume uptake was measured in milligrams. For DNA release various washing out buffers were used and the amount of released DNA was measured spectrophotometrically. The amount of protein released from the swabs were examined using the Lowry assay. We observed statistically significant differences (p < 0.05) in the mean weights of absorbed liquid, in the DNA recovery and protein recovery by the four variety of swab examined. However, the efficiency of DNA and protein release was not correlated to the absorbed volume of a sample, but rather to the properties of swabs. The swab composition and structure can have a significant impact on the collection and release efficiency of a sample. Therefore, validation of POCT in relation to the used swabs for sampling is really important. The use of inappropriate swabs could lead to false negative or misleading analysis results.

In situ antibody-loaded hydrogel for intravitreal delivery

Therapeutic protein medicines have transformed the treatment of blinding diseases (e.g. age-related macular degeneration, AMD) during the last 1–2 decades. Many blinding conditions such as AMD are chronic; and require multiple intravitreal injections over a long period to achieve a high and reproducible dose needed for clinical benefit. Prolonging the duration of action of ophthalmic drugs is critical to reduce the frequency of injections. Thermoresponsive hydrogels (e.g. N-isopropylacrylamide, NIPAAM) that collapse in physiological conditions can entrap and sustain the release of a therapeutic protein. However, most NIPAAM hydrogels are not biodegradable and often requires invasive surgery to remove the depot. Here, we report the preparation of a hydrogel derived from NIPAAM and acrylated hyaluronic acid (Ac-HA) as a biodegradable, macromolecular crosslinker. Ac-HA was prepared by the acrylation of hyaluronic acid (HA). Antibody (infliximab (INF), 5.0 mg/mL or bevacizumab (BEVA), 12.5 mg/mL), NIPAAM (0.35 mmol) and Ac-HA (2.0–10.0 mg/mL, 40.0–200.0 nmol) were first mixed prior to redox polymerisation to ensure maximal protein mixing and to shorten the burst release. Hydrogels with lower amounts of Ac-HA (2.0–4.0 mg/mL, 40.0–80.0 nmol) showed favourable lower critical solution temperature (LCST) values and injectability (27–29G) than higher amounts of Ac-HA (>4.0 mg/mL, >80.0 nmol). These hydrogels were further characterised (swelling ratio (SR), water retention (WR) and rheology). All hydrogels degraded in presence of bovine testes hyaluronidase (0–50 U/mL, 37 °C, 100 rpm). Release studies of BEVA-loaded hydrogels were investigated in vitro using the PK-Eye™ model, which estimates the human clearance times of proteins from the back of the eye. Phosphate buffered saline (PBS, pH 7.4, 37 °C) was used rather than simulated vitreous to more effectively map trends between the formulations. A zero-order release profile was observed between days 5 to 50 with 43.3 ± 9.5% protein released at day 50. Determining protein binding and functionality from a formulation is crucial to determine the optimal formulation prior to more detailed studies that might be necessary. BEVA showed binding to human vascular growth endothelial factor (VEGF165) throughout the study (two months) while still maintaining a therapeutic dose (123.5 ± 45.6 ng) in the posterior cavity of the PK-Eye™ model. These encouraging results suggest that extended release of proteins in the vitreous can be achieved using injectable hydrogels derived from NIPAAM and HA.

Comparison of Four Quantitative Techniques for Monitoring Microalgae Disruption by Low-Frequency Ultrasound and Acoustic Energy Efficiency.

Ultrasound has been regarded as an environmental friendly technology to utilize microalgae biomass and control algal blooms. In this study, four quantitative techniques, including cell counting, optical density of algal suspension, pigments release, and protein release, were performed on three species of microalgae ( M.aeruginosa, C.pyrenoidosa, and C.reinhardtii) to develop effective techniques for rapid monitoring of cell disruption and to optimize the acoustic energy efficiency. Results showed optical density of algal suspensions was not an optimal indicator to monitor cell disruption, although it is a common technique for determining cell concentration in microbial cultures. Instead, an accurate and reliable technique was to determine the release of intracellular pigments (absorbance peaks of supernatant) for indicating cell rupture. The protein released during sonication could also be a useful indicator if it is the component of interest. A fitted power functional model showed a strong relationship between cell disruption and energy consumption ( R2 > 0.87). This model could provide an effective approach to directly compare the energy efficiency of ultrasound in different systems or with varying microalgae species. This study provides valuable information for microalgae utilization and the treatment of algal blooms by ultrasound, so as to achieve energy conservation and environmental safety.

Hydrogels based on copolymers of 2‐hydroxyethylmethacrylate and 2‐hydroxyethylacrylate as a delivery system for proteins: Interactions with lysozyme

ABSTRACTHydrogels have attracted considerable attention due to numerous applications, in particular as contact lenses and carriers for sustained drug delivery. The aim of the present work is to characterize the interactions of copolymer hydrogels consisted of 2‐hydroxyethylmethacrylate (HEMA) and 2‐hydroxyethylacrylate (HEA) with a small protein (lysozyme) and to assess the potential applications of these hydrogels as a drug delivery system for sustained release of protein‐based therapeutics. Physicochemical properties of protein‐loaded hydrogels, as well as lysozyme in vitro loading and release and the conformation of the protein released from hydrogels were studied. The effect of copolymer composition on the protein deposition on hydrogels and protein aggregation in the presence of hydrogels was also assessed. The results show that introduction of HEA into the copolymeric hydrogels enhances their suitability as a delivery system for proteins. Copolymerisation of HEMA and HEA allows controlling the physicochemical properties of hydrogels and the protein release rate. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44768.

Protective immunity against toxoplasmosis in mice induced by single-dose immunization with rSAG1/2 protein released from poly(lactide-co-glycolide) microparticles.

Triphasic sustained release of tachyzoite chimeric protein, rSAG1/2, from poly(lactide-co-glycolide) (PLG)-encapsulated rSAG1/2 (PLG-rSAG1/2) microparticles (MPs) is a promising characteristic for developing a single-dose vaccine against Toxoplasma gondii in domestic animals. In the present study, we aimed to evaluate whether single immunization with PLG-rSAG1/2 MPs in BALB/c mice would achieve effective immunity and protection against T. gondii. Peritoneal immunization of mice with a single dose of PLG-rSAG1/2 MPs enhanced serum IgG titers and lymphocyte proliferation in a triphasic model over a long 12-week period. In addition, 12 weeks after immunization, significant production of IFN-γ was also monitored in mice vaccinated with one dose of PLG-rSAG1/2 MPs. More importantly, the immunity induced by one dose of PLG-rSAG1/2 MPs protected 70% of mice (14/20) against a lethal subcutaneous challenge of 1 × 104 live tachyzoites of T. gondii (RH strain). In conclusion, a single dose of PLG-rSAG1/2 MPs capable of sustaining triphasic release of rSAG1/2 protein induces long-lasting triphasic immunity against T. gondii in mice. Our data indicate the feasibility of PLG-rSAG1/2 MPs to be developed as a single-dose vaccine against T. gondii for potential use in domestic animals.

RANKL release from self-assembling nanofiber hydrogels for inducing osteoclastogenesis in vitro.

To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL. Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala)4-CONH2 [(RADA)4] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers. NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL. This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures. Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth, facilitating the required changes in the surrounding alveolar bone through the process of bone remodelling. The RANKL system regulates alveolar bone remodelling and controls root resorption during OTM. The use of exogenous RANKL to accelerate OTM has not been attempted to date because large quantities of RANKL for systemic therapy may subsequently cause serious systemic loss of skeletal bone. The controlled and sustained local release of RANKL from a carrier matrix could maximize its therapeutic benefit whilst minimizing systemic side effects. In this study a NF-hydrogel was used for sustained and controlled release of RANKL and the release kinetics and biofunctionality of the released RANKL was characterized. Our results provide fundamental insight for further investigating the role of RANKL NF-hydrogel release systems for inducing osteoclastogenesis in vivo.

Human serum albumin in electrospun PCL fibers: structure, release, and exposure on fiber surface

Human serum albumin (HSA) introduced to the fibers produced by electrospinning from HSA and polycaprolactone (PCL) solutions in hexafluoroisopropanol has been studied in terms of its structure, release from the fibers, stability of interaction with basic polymer, accessibility for protease attack, and cellular receptors, as well as dependence of the studied parameters on the protein concentration in fibers. A limited part of the protein leaves the fibers right after soaking with water, whereas the remaining protein stays tightly bound to fibers for a long time because protein nanoparticles are tightly integrated with PCL, as shown by small-angle X-ray scattering. As has been demonstrated, the proteins leave the fibers in complexes with PCL. X-ray photoelectron spectroscopy demonstrates that the protein concentration on the fiber surface is higher than the concentration in electrospinning solution. The surface-exposed protein is recognized by cell receptors and is partially hydrolyzed by proteinase K. The data on pulse protein release, presence of PCL in the protein released from matrixes, overrepresentation of the protein on the fiber surface, and tight interaction of protein with PCL may be useful for rational design of electrospun scaffolds intended for drug delivery and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Controlled release of NELL-1 protein from chitosan/hydroxyapatite-modified TCP particles

NEL-like molecule-1 (NELL-1) is a novel osteogenic protein that showing high specificity to osteochondral cells. It was widely used in bone regeneration research by loading onto carriers such as tricalcium phosphate (TCP) particles. However, there has been little research on protein controlled release from this material and its potential application. In this study, TCP was first modified with a hydroxyapatite coating followed by a chitosan coating to prepare chitosan/hydroxyapatite-coated TCP particles (Chi/HA-TCP). The preparation was characterized by SEM, EDX, FTIR, XRD, FM and Zeta potential measurements. The NELL-1 loaded Chi/HA-TCP particles and the release kinetics were investigated in vitro. It was observed that the Chi/HA-TCP particles prepared with the 0.3% (wt/wt) chitosan solution were able to successfully control the release of NELL-1 and maintain a slow, steady release for up to 28 days. Furthermore, more than 78% of the loaded protein⿿s bioactivity was preserved in Chi/HA-TCP particles over the period of the investigation, which was significantly higher than that of the protein released from hydroxyapatite coated TCP (HA-TCP) particles. Collectively, this study suggests that the osteogenic protein NELL-1 showed a sustained release pattern after being encapsulated into the modified Chi/HA-TCP particles, and the NELL-1 integrated composite of Chi/HA-TCP showed a potential to function as a protein delivery carrier and as an improved bone matrix for use in bone regeneration research.

P253Oxidative stress causes the release of a specific subset of proteins from viable cardiac myocytes

Purpose: It is widely accepted that oxidative stress plays a major role in cardiovascular diseases. The aim of this study is to identify novel biomarkers that are released from cardiomyocytes upon oxidative stress before the onset of necrosis. Methods and Results: Firstly, isolated mouse hearts were subjected to Langendorff perfusion with 1 mM H2O2 for 15 min to induce maximal oxidative stress in the absence of cell death, as detected by lactate dehydrogenase release. Notably, SDS/PAGE of tissue samples displayed the disappearance of a band migrating at 72 kDa in the H2O2 group as compared to the normoxic control. Analysis by mass spectrometry identified two proteins, namely EHD4 and HSPA8. Concomitantly, these proteins were found in the coronary effluent. We confirmed this result by both Western blot and immunohistochemistry. In particular, H2O2-perfused heart cryosections displayed 77% and 70% decrease in immunofluorescence as compared to normoxic-perfused hearts for EDH4 and HSPA8, respectively. EHD4 belongs to the EHD protein family that are involved in intracellular trafficking, while HSPA8 is a chaperonin. Then, we evaluated another model of oxidative stress caused by single injection of doxorubicin (15 mg/kg). Mice were sacrificed after 3 days. The EHD4 content was reduced in hearts of doxorubicin-treated mice, as compared to hearts of vehicle-treated animals. To confirm that protein release can occur the absence of cell death, we treated an atriomal murine cell line (HL1) with: H2O2 (10-100 μM), doxorubicin (1-6 μM), or H2O2 (10 μM) + doxorubicin (6 μM). We observed that the intracellular EHD4 content was significantly reduced upon the different treatments, as detected by Western blot and immunocytochemistry in the absence of any loss of cell viability, as detected by Hoecst 22358/Propidium Iodide staining. The release of HSPA8 in the tested protocols was less evident. Conclusions: EHD4 appears as the most prominent protein released by cardiac myocytes undergoing oxidative stress in the absence of cell death. Therefore, EHD4 release should be exploited as a novel plasma biomarker of early oxidative stress, occurring before the onset of necrosis. Further studies will elucidate the mechanisms underlying protein release in viable cardiac myocytes and the effects of specific protein loss on the susceptibility to cell injury.

Strategies for neurotrophin-3 and chondroitinase ABC release from freeze-cast chitosan-alginate nerve-guidance scaffolds.

Freeze casting, or controlled unidirectional solidification, can be used to fabricate chitosan-alginate (C-A) scaffolds with highly aligned porosity that are suitable for use as nerve-guidance channels. To augment the guidance of growth across a spinal cord injury lesion, these scaffolds are now evaluated in vitro to assess their ability to release neurotrophin-3 (NT-3) and chondroitinase ABC (chABC) in a controlled manner. Protein-loaded microcapsules were incorporated into C-A scaffolds prior to freeze casting without affecting the original scaffold architecture. In vitro protein release was not significantly different when comparing protein loaded directly into the scaffolds with release from scaffolds containing incorporated microcapsules. NT-3 was released from the C-A scaffolds for 8 weeks in vitro, while chABC was released for up to 7 weeks. Low total percentages of protein released from the scaffolds over this time period were attributed to limitation of diffusion by the interpenetrating polymer network matrix of the scaffold walls. NT-3 and chABC released from the scaffolds retained bioactivity, as determined by a neurite outgrowth assay, and the promotion of neurite growth across an inhibitory barrier of chondroitin sulphate proteoglycans. This demonstrates the potential of these multifunctional scaffolds for enhancing axonal regeneration through growth-inhibiting glial scars via the sustained release of chABC and NT-3. Copyright © 2014 John Wiley & Sons, Ltd.

The Effects of High Intensity Short Rest Resistance Exercise on Muscle Damage Markers in Men and Women

Within and between sexes, universal load prescription (as assigned in extreme conditioning programs) creates extreme ranges in individual training intensities. Exercise intensity has been proposed to be the main factor determining the degree of muscle damage. Thus, the purpose of this study was to examine markers of muscle damage in resistance-trained men (n = 9) and women (n = 9) from a high intensity (HI) short rest (SR) (HI/SR) resistance exercise protocol. The HI/SR consisted of a descending pyramid scheme starting at 10 repetitions, decreasing 1 repetition per set for the back squat, bench press, and deadlift, as fast as possible. Blood was drawn pre-exercise (pre), immediately postexercise (IP), 15 minutes postexercise (+15), 60 minutes postexercise (+60), and 24 hours postexercise (+24). Women demonstrated significant increases in interleukin 6 (IL-6; IP), creatine kinase (CK; +24), myoglobin (IP, +15, +60), and a greater relative increase when compared with men (+15, +60). Men demonstrated significant increases in myoglobin (IP, +15, +60, +24), IL-6 (IP, +15), CK (IP, +60, +24), and testosterone (IP, +15). There were significant sex interactions observed in CK (IP, +60, +24) and testosterone (IP, +15, +60, +24). Women completed the protocol faster (women: 34:04 ± 9:40 minutes, men: 39:22 ± 14:43 minutes), and at a slightly higher intensity (women: 70.1 ± 3.5%, men 68.8 ± 3.1%); however, men performed significantly more work (men: 14384.6 ± 1854.5 kg, women: 8774.7 ± 1612.7 kg). Overall, women demonstrated a faster inflammatory response with increased acute damage, whereas men demonstrated a greater prolonged damage response. Therefore, strength and conditioning professionals need to be aware of the level of stress imposed on individuals when creating such volitional high intensity metabolic type workouts and allow for adequate progression and recovery from such workouts.

Yeast: A Potential Biomass Substrate for the Production of Cleaner Energy (Biogas)

Yeast cell wall and its entire contents disruption treatments are required in the enhancement of protein and the overall biodegradability of the cell wall materials during homogenization process. Yeast as a cheap, good resource and easily available source of energy from biomass into biogas, it was used as a substrate for the cleaner energy production due to its richly and high level content of protein contained in it.An initial study on the effects of high-pressure homogenizer mechanical pretreatment has been conducted in sequence as generated by the design matrix of the design of experiment (DOE) focusing on protein yields from bakers’ yeast also known as Saccharomyces cerevisiae and in order to achieve the maximum yield of protein which in other words aid biogas production, the following optimum process parameters were set in. The yeast block was refrigerated at between 0 – 4°C with fermentation at (0 – 24h), a pH value of (5.3) maximum was used in the preparation of the buffer solution C. This was obtained through diluting solution B into A until the pH was attained (details as shown in the materials and methods section). Number of cycles (passes) of the soluble yeast were undergone to enable the yeast cell walls be broken down for the release of more protein and at temperature range (15 – 25°C). The pressure for the compressed state during homogenization was set between (30 – 90MPa). The results presented therefore showed the rates of protein released from the disruption through using the Design Expert Software V.8 in identifying the ideal conditions as set in the parameters.