Cyclic Mechanical Loading Enhances Transport of Antibodies Into Articular Cartilage.

PubMed

DiDomenico, Chris D; Xiang Wang, Zhen; Bonassar, Lawrence J

2017-01-01

The goal of this study was to characterize antibody penetration through cartilage tissue under mechanical loading. Mechanical stimulation aids in the penetration of some proteins, but this effect has not characterized molecules such as antibodies (>100 kDa), which may hold some clinical value for treating osteoarthritis (OA). For each experiment, fresh articular cartilage plugs were obtained and exposed to fluorescently labeled antibodies while under cyclic mechanical load in unconfined compression for several hours. Penetration of these antibodies was quantified using confocal microscopy, and finite element (FE) simulations were conducted to predict fluid flow patterns within loaded samples. Transport enhancement followed a linear trend with strain amplitude (0.25-5%) and a nonlinear trend with frequency (0.25-2.60 Hz), with maximum enhancement found to be at 5% cyclic strain and 1 Hz, respectively. Regions of highest enhancement of transport within the tissue were associated with the regions of highest interstitial fluid velocity, as predicted from finite-element simulations. Overall, cyclic compression-enhanced antibody transport by twofold to threefold. To our knowledge, this is the first study to test how mechanical stimulation affects the diffusion of antibodies in cartilage and suggest further study into other important factors regarding macromolecular transport.

Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

PubMed

Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

2017-05-15

Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Voluntary resistance running with short distance enhances spatial memory related to hippocampal BDNF signaling.

PubMed

Lee, Min Chul; Okamoto, Masahiro; Liu, Yu Fan; Inoue, Koshiro; Matsui, Takashi; Nogami, Haruo; Soya, Hideaki

2012-10-15

Although voluntary running has beneficial effects on hippocampal cognitive functions if done abundantly, it is still uncertain whether resistance running would be the same. For this purpose, voluntary resistance wheel running (RWR) with a load is a suitable model, since it allows increased work levels and resultant muscular adaptation in fast-twitch muscle. Here, we examined whether RWR would have potential effects on hippocampal cognitive functions with enhanced hippocampal brain-derived neurotrophic factor (BDNF), as does wheel running without a load (WR). Ten-week-old male Wistar rats were assigned randomly to sedentary (Sed), WR, and RWR (to a maximum load of 30% of body weight) groups for 4 wk. We found that in RWR, work levels increased with load, but running distance decreased by about half, which elicited muscular adaptation for fast-twitch plantaris muscle without causing any negative stress effects. Both RWR and WR led to improved spatial learning and memory as well as gene expressions of hippocampal BDNF signaling-related molecules. RWR increased hippocampal BDNF, tyrosine-related kinase B (TrkB), and cAMP response element-binding (CREB) protein levels, whereas WR increased only BDNF. With both exercise groups, there were correlations between spatial memory and BDNF protein (r = 0.41), p-CREB protein (r = 0.44), and work levels (r = 0.77). These results suggest that RWR plays a beneficial role in hippocampus-related cognitive functions associated with hippocampal BDNF signaling, even with short distances, and that work levels rather than running distance are more determinant of exercise-induced beneficial effects in wheel running with and without a load.

Akt1 deficiency diminishes skeletal muscle hypertrophy by reducing satellite cell proliferation.

PubMed

Moriya, Nobuki; Miyazaki, Mitsunori

2018-05-01

Skeletal muscle mass is determined by the net dynamic balance between protein synthesis and degradation. Although the Akt/mechanistic target of rapamycin (mTOR)-dependent pathway plays an important role in promoting protein synthesis and subsequent skeletal muscle hypertrophy, the precise molecular regulation of mTOR activity by the upstream protein kinase Akt is largely unknown. In addition, the activation of satellite cells has been indicated as a key regulator of muscle mass. However, the requirement of satellite cells for load-induced skeletal muscle hypertrophy is still under intense debate. In this study, female germline Akt1 knockout (KO) mice were used to examine whether Akt1 deficiency attenuates load-induced skeletal muscle hypertrophy through suppressing mTOR-dependent signaling and satellite cell proliferation. Akt1 KO mice showed a blunted hypertrophic response of skeletal muscle, with a diminished rate of satellite cell proliferation following mechanical overload. In contrast, Akt1 deficiency did not affect the load-induced activation of mTOR signaling and the subsequent enhanced rate of protein synthesis in skeletal muscle. These observations suggest that the load-induced activation of mTOR signaling occurs independently of Akt1 regulation and that Akt1 plays a critical role in regulating satellite cell proliferation during load-induced muscle hypertrophy.

Surface enhanced fluorescence of anti-tumoral drug emodin adsorbed on silver nanoparticles and loaded on porous silicon

NASA Astrophysics Data System (ADS)

Hernandez, Margarita; Recio, Gonzalo; Martin-Palma, Raul J.; Garcia-Ramos, Jose V.; Domingo, Concepcion; Sevilla, Paz

2012-07-01

Fluorescence spectra of anti-tumoral drug emodin loaded on nanostructured porous silicon have been recorded. The use of colloidal nanoparticles allowed embedding of the drug without previous porous silicon functionalization and leads to the observation of an enhancement of fluorescence of the drug. Mean pore size of porous silicon matrices was 60 nm, while silver nanoparticles mean diameter was 50 nm. Atmospheric and vacuum conditions at room temperature were used to infiltrate emodin-silver nanoparticles complexes into porous silicon matrices. The drug was loaded after adsorption on metal surface, alone, and bound to bovine serum albumin. Methanol and water were used as solvents. Spectra with 1 μm spatial resolution of cross-section of porous silicon layers were recorded to observe the penetration of the drug. A maximum fluorescence enhancement factor of 24 was obtained when protein was loaded bound to albumin, and atmospheric conditions of inclusion were used. A better penetration was obtained using methanol as solvent when comparing with water. Complexes of emodin remain loaded for 30 days after preparation without an apparent degradation of the drug, although a decrease in the enhancement factor is observed. The study reported here constitutes the basis for designing a new drug delivery system with future applications in medicine and pharmacy.

Effects of hydrogenated TiO2 nanotube arrays on protein adsorption and compatibility with osteoblast-like cells.

PubMed

Lu, Ran; Wang, Caiyun; Wang, Xin; Wang, Yuji; Wang, Na; Chou, Joshua; Li, Tao; Zhang, Zhenting; Ling, Yunhan; Chen, Su

2018-01-01

Modified titanium (Ti) substrates with titanium dioxide (TiO 2 ) nanotubes have broad usage as implant surface treatments and as drug delivery systems. To improve drug-loading capacity and accelerate bone integration with titanium, in this study, we hydrogenated anodized titanium dioxide nanotubes (TNTs) by a thermal treatment. Three groups were examined, namely: hydrogenated TNTs (H 2 -TNTs, test), unmodified TNTs (air-TNTs, control), and Ti substrates (Ti, control). Our results showed that oxygen vacancies were present in all the nanotubes. The quantity of -OH groups greatly increased after hydrogenation. Furthermore, the protein adsorption and loading capacity of the H 2 -TNTs were considerably enhanced as compared with the properties of the air-TNTs ( P <0.05). Additionally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to investigate the interactions of TNTs with proteins. During the protein-loading process, the H 2 -TNTs not only enabled rapid protein adsorption, but also decreased the rate of protein elution compared with that of the air-TNTs. We found that the H 2 -TNTs exhibited better biocompatibility than the air-TNT and Ti groups. Both cell adhesion activity and alkaline phosphatase activity were significantly improved toward MG-63 human osteoblast-like cells as compared with the control groups ( P <0.05). We conclude that hydrogenated TNTs could greatly improve the loading capacity of bioactive molecules and MG-63 cell proliferation.

The effects of dynamic compressive loading on biodegradable implants of 50-50% polylactic Acid-polyglycolic Acid.

PubMed

Thompson, D E; Agrawal, C M; Athanasiou, K

1996-01-01

Biodegradable implants that release growth factors or other bioactive agents in a controlled manner are investigated to enhance the repair of musculoskeletal tissues. In this study, the in vitro release characteristics and mechanical properties of a 50:50 polylactic acid/polyglycolic acid two phase implant were examined over a 6-week period under no-load conditions or under a cyclic compressive load, such as that experienced when walking slowly during rehabilitation. The results demonstrated that a cyclic compressive load significantly slows the decrease of molecular chain size during the first week, significantly increases protein release for the first 2-3 weeks, and significantly stiffens the implant for the first 3 weeks. It was also shown that protein release is initially high and steadily decreases with time until the molecular weight declines to about 20% of its original value (approximately 4 weeks). Once this threshold is reached, increased protein release, surface deformation, and mass loss occurs. This study also showed that dynamic loading and the environment in which an implant is placed affect its biodegradation. Therefore, it may be essential that in vitro degradation studies of these or similar implants include a dynamic functional environment.

Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics

NASA Astrophysics Data System (ADS)

Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Sankar Ghosh, Siddhartha

2014-08-01

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics.

PubMed

Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Ghosh, Siddhartha Sankar

2014-08-29

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a 'green synthesis' method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

New valve and bonding designs for microfluidic biochips containing proteins.

PubMed

Lu, Chunmeng; Xie, Yubing; Yang, Yong; Cheng, Mark M-C; Koh, Chee-Guan; Bai, Yunling; Lee, L James; Juang, Yi-Je

2007-02-01

Two major concerns in the design and fabrication of microfluidic biochips are protein binding on the channel surface and protein denaturing during device assembly. In this paper, we describe new methods to solve these problems. A "fishbone" microvalve design based on the concept of superhydrophobicity was developed to replace the capillary valve in applications where the chip surface requires protein blocking to prevent nonspecific binding. Our experimental results show that the valve functions well in a CD-like ELISA device. The packaging of biochips containing pre-loaded proteins is also a challenging task since conventional sealing methods often require the use of high temperatures, electric voltages, or organic solvents that are detrimental to the protein activity. Using CO2 gas to enhance the diffusion of polymer molecules near the device surface can result in good bonding at low temperatures and low pressure. This bonding method has little influence on the activity of the pre-loaded proteins after bonding.

Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

PubMed

Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

2015-06-01

Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

Derivatized graphitic nanofibres (GNF) as a new support material for mass spectrometric analysis of peptides and proteins.

PubMed

Greiderer, Andreas; Rainer, Matthias; Najam-ul-Haq, Muhammad; Vallant, Rainer M; Huck, Christian W; Bonn, Günther K

2009-07-01

Graphitic nanofibres (GNFs), 100-200 nm in diameter and 5-20 microm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 microg ml(-1) for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol microl(-1) (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.

Management of Natural and Added Dietary Phosphorus Burden in Kidney Disease

PubMed Central

Cupisti, Adamasco; Kalantar-Zadeh, Kamyar

2018-01-01

Phosphorus retention occurs from higher dietary phosphorus intake relative to its renal excretion or dialysis removal. In the gastrointestinal tract the naturally existing organic phosphorus is only partially (~60%) absorbable; however, this absorption varies widely and is lower for plant-based phosphorus including phytate (<40%) and higher for foods enhanced with inorganic-phosphorus-containing preservatives (>80%). The latter phosphorus often remains unrecognized by patients and health care professionals, even though it is widely used in contemporary diets, in particular low-cost foods. In a non-enhanced mixed diet, the digestible phosphorus is closely correlated with total protein content, making protein-rich foods a main source of natural phosphorus. Phosphorus burden is more appropriately limited in pre-dialysis patients who are on low protein diets (~0.6 g/kg/day), whereas dialysis patients who require higher protein intake (~1.2 g/kg/day) are subject to a higher dietary phosphorus load. An effective and patient-friendly approach to reduce phosphorus intake without depriving patients of adequate proteins is to educate patients to avoid foods with high phosphorus relative to protein such as egg yolk and those with high amounts of phosphorus-based preservatives such as certain soft drinks and enhanced cheese and meat. Protein-rich foods should be prepared by boiling, which reduces phosphorus as well as sodium and potassium content, or by other types of cooking induced demineralization. The dose of phosphorus-binding therapy should be adjusted separately for the amount and absorbability of phosphorus in each meal. Dietician counselling to address the foregoing aspects of dietary phosphorus management is instrumental for achieving reduction of phosphorus load. PMID:23465504

Promotion of the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles combined with polypropylene electret.

PubMed

Tu, Ye; Wang, Xinxia; Lu, Ying; Zhang, He; Yu, Yuan; Chen, Yan; Liu, Junjie; Sun, Zhiguo; Cui, Lili; Gao, Jing; Zhong, Yanqiang

We recently reported that electret, which was prepared by a corona charging system with polypropylene film, could enhance the transdermal delivery of several drugs of low molecular weight. The aim of this study was to investigate whether electret could enhance the transdermal delivery of protein drugs by N -trimethyl chitosan nanoparticles (TMC NPs) prepared by an ionic gelation method. A series of experiments were performed, including in vitro skin permeation assays and anti-inflammatory effects, to evaluate the transdermal delivery of protein drugs by TMC NPs in the presence of electret. The results showed that in the presence of electret, the transdermal delivery of protein drugs in TMC NPs was significantly enhanced, as demonstrated by in vitro permeation studies and confocal laser scanning microscopy. Notably, superoxide dismutase-loaded TMC NPs combined with electret exhibited the best inhibitory effect on the edema of the mouse ear. TMC NPs combined with electret represent a novel platform for the transdermal delivery of protein drugs.

Promotion of the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles combined with polypropylene electret

PubMed Central

Tu, Ye; Wang, Xinxia; Lu, Ying; Zhang, He; Yu, Yuan; Chen, Yan; Liu, Junjie; Sun, Zhiguo; Cui, Lili; Gao, Jing; Zhong, Yanqiang

2016-01-01

We recently reported that electret, which was prepared by a corona charging system with polypropylene film, could enhance the transdermal delivery of several drugs of low molecular weight. The aim of this study was to investigate whether electret could enhance the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles (TMC NPs) prepared by an ionic gelation method. A series of experiments were performed, including in vitro skin permeation assays and anti-inflammatory effects, to evaluate the transdermal delivery of protein drugs by TMC NPs in the presence of electret. The results showed that in the presence of electret, the transdermal delivery of protein drugs in TMC NPs was significantly enhanced, as demonstrated by in vitro permeation studies and confocal laser scanning microscopy. Notably, superoxide dismutase-loaded TMC NPs combined with electret exhibited the best inhibitory effect on the edema of the mouse ear. TMC NPs combined with electret represent a novel platform for the transdermal delivery of protein drugs. PMID:27822034

A ToF-SIMS and XPS study of protein adsorption and cell attachment across PEG-like plasma polymer films with lateral compositional gradients

NASA Astrophysics Data System (ADS)

Menzies, Donna J.; Jasieniak, Marek; Griesser, Hans J.; Forsythe, John S.; Johnson, Graham; McFarland, Gail A.; Muir, Benjamin W.

2012-12-01

In this work we report a detailed X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) study of poly(ethylene glycol) PEG-like chemical gradients deposited via plasma enhanced chemical vapour deposition (PECVD) at two different load powers using diethylene glycol dimethyl ether (DG) as a monomer. Principal component analysis (PCA) was applied to the ToF-SIMS data both before and after protein adsorption on the plasma polymer thin films. Results of the PCA loadings indicated a higher content of hydrocarbon fragments across the higher load power gradient, which adsorbed higher amounts of proteins. Gradients deposited at a lower load power retained a higher degree of monomer like functionality as did the central region directly underneath the knife edge electrode. Analysis of the adsorption of serum proteins (human serum albumin and fetal bovine serum) was monitored across the gradient films and increased with decreasing ether (PEG-like) film chemistries. The effect of protein incubation time on the levels adsorbed fetal bovine serum on the plasma polymer films was critical, with significantly more protein adsorbing after 24 hour incubation times on both gradient films. The attachment of HeLa cells on the gradients appeared to be dictated not only by the surface chemistry, but also by the adsorption of serum proteins. XPS analysis revealed that at surface ether concentrations of less than 70% in the gradient films, significant increases in protein and cell attachment were observed.

Transdermal delivery of biomacromolecules using lipid-like nanoparticles

NASA Astrophysics Data System (ADS)

Bello, Evelyn A.

The transdermal delivery of biomacromolecules, including proteins and nucleic acids, is challenging, owing to their large size and the penetration-resistant nature of the stratum corneum. Thus, an urgent need exists for the development of transdermal delivery methodologies. This research focuses on the use of cationic lipid-like nanoparticles (lipidoids) for the transdermal delivery of proteins, and establishes an in vitro model for the study. The lipidoids used were first combinatorially designed and synthesized; afterwards, they were employed for protein encapsulation in a vesicular system. A skin penetration study demonstrated that lipidoids enhance penetration depth in a pig skin model, overcoming the barrier that the stratum corneum presents. This research has successfully identified active lipidoids capable of efficiently penetrating the skin; therefore, loading proteins into lipidoid nanoparticles will facilitate the transdermal delivery of proteins. Membrane diffusion experiments were used to confirm the results. This research has confirmed that lipidoids are a suitable material for transdermal protein delivery enhancement.

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing.

PubMed

Schrier, J A; Fink, B F; Rodgers, J B; Vasconez, H C; DeLuca, P P

2001-10-07

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization, matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well, allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days, with the unembedded microspheres releasing faster than those embedded in CMC. In vivo, the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2/PLGA microspheres in CMC was an effective implantable protein-delivery system for use in bone repair.

Targeted intracellular delivery of proteins with spatial and temporal control.

PubMed

Morales, Demosthenes P; Braun, Gary B; Pallaoro, Alessia; Chen, Renwei; Huang, Xiao; Zasadzinski, Joseph A; Reich, Norbert O

2015-02-02

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model.

PubMed

Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

2017-02-10

In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease.

Biologically tunable reactivity of energetic nanomaterials using protein cages.

PubMed

Slocik, Joseph M; Crouse, Christopher A; Spowart, Jonathan E; Naik, Rajesh R

2013-06-12

The performance of aluminum nanomaterial based energetic formulations is dependent on the mass transport, diffusion distance, and stability of reactive components. Here we use a biologically inspired approach to direct the assembly of oxidizer loaded protein cages onto the surface of aluminum nanoparticles to improve reaction kinetics by reducing the diffusion distance between the reactants. Ferritin protein cages were loaded with ammonium perchlorate (AP) or iron oxide and assembled with nAl to create an oxidation-reduction based energetic reaction and the first demonstration of a nanoscale biobased thermite material. Both materials showed enhanced exothermic behavior in comparison to nanothermite mixtures of bulk free AP or synthesized iron oxide nanopowders prepared without the use of ferritin. In addition, by utilizing a layer-by-layer (LbL) process to build multiple layers of protein cages containing iron oxide and iron oxide/AP on nAl, stoichiometric conditions and energetic performance can be optimized.

Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response

PubMed Central

Goldstein, Ido; Baek, Songjoon; Presman, Diego M.; Paakinaho, Ville; Swinstead, Erin E.; Hager, Gordon L.

2017-01-01

Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting. PMID:28031249

Delivery of Dual Drug Loaded Lipid Based Nanoparticles across the Blood-Brain Barrier Impart Enhanced Neuroprotection in a Rotenone Induced Mouse Model of Parkinson's Disease.

PubMed

Kundu, Paromita; Das, Manasi; Tripathy, Kalpalata; Sahoo, Sanjeeb K

2016-12-21

Parkinson's disease (PD) is the most widespread form of dementia where there is an age related degeneration of dopaminergic neurons in the substantia nigra region of the brain. Accumulation of α-synuclein (αS) protein aggregate, mitochondrial dysfunction, oxidative stress, and neuronal cell death are the pathological hallmarks of PD. In this context, amalgamation of curcumin and piperine having profound cognitive properties, and antioxidant activity seems beneficial. However, the blood-brain barrier (BBB) is the major impediment for delivery of neurotherapeutics to the brain. The present study involves formulation of curcumin and piperine coloaded glyceryl monooleate (GMO) nanoparticles coated with various surfactants with a view to enhance the bioavailability of curcumin and penetration of both drugs to the brain tissue crossing the BBB and to enhance the anti-parkinsonism effect of both drugs in a single platform. In vitro results demonstrated augmented inhibition of αS protein into oligomers and fibrils, reduced rotenone induced toxicity, oxidative stress, and apoptosis, and activation of autophagic pathway by dual drug loaded NPs compared to native counterpart. Further, in vivo studies revealed that our formulated dual drug loaded NPs were able to cross BBB, rescued the rotenone induced motor coordination impairment, and restrained dopaminergic neuronal degeneration in a PD mouse model.

Modulation of Stromal Cell-Derived Factor-1/CXC Chemokine Receptor 4 Axis Enhances rhBMP-2-Induced Ectopic Bone Formation

PubMed Central

Wise, Joel K.; Sumner, Dale Rick

2012-01-01

Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation. PMID:22035136

Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robling, Alexander G.; Kang, Kyung Shin; Bullock, Whitney A.

Here, sclerostin ( Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sostmore » –/– mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous h SOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5–/– mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost–/– mice, which are resistant to disuse-induced bone loss, ECR5–/– mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.« less

Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation

DOE PAGES

Robling, Alexander G.; Kang, Kyung Shin; Bullock, Whitney A.; ...

2016-09-04

Here, sclerostin ( Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sostmore » –/– mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous h SOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5–/– mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost–/– mice, which are resistant to disuse-induced bone loss, ECR5–/– mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.« less

Synthesis of protein-coated biocompatible methotrexate-loaded PLA-PEG-PLA nanoparticles for breast cancer treatment

PubMed Central

Massadeh, Salam; Alaamery, Manal; Al-Qatanani, Shatha; Alarifi, Saqer; Bawazeer, Shahad; Alyafee, Yusra

2016-01-01

Background PLA-PEG-PLA triblock polymer nanoparticles are promising tools for targeted dug delivery. The main aim in designing polymeric nanoparticles for drug delivery is achieving a controlled and targeted release of a specific drug at the therapeutically optimal rate and choosing a suitable preparation method to encapsulate the drug efficiently, which depends mainly on the nature of the drug (hydrophilic or hydrophobic). In this study, methotrexate (MTX)-loaded nanoparticles were prepared by the double emulsion method. Method Biodegradable polymer polyethylene glycol-polylactide acid tri-block was used with poly(vinyl alcohol) as emulsifier. The resulting methotrexate polymer nanoparticles were coated with bovine serum albumin in order to improve their biocompatibility. This study focused on particle size distribution, zeta potential, encapsulation efficiency, loading capacity, and in vitro drug release at various concentrations of PVA (0.5%, 1%, 2%, and 3%). Results Reduced particle size of methotrexate-loaded nanoparticles was obtained using lower PVA concentrations. Enhanced encapsulation efficiency and loading capacity was obtained using 1% PVA. FT-IR characterization was conducted for the void polymer nanoparticles and for drug-loaded nanoparticles with methotrexate, and the protein-coated nanoparticles in solid state showed the structure of the plain PEG-PLA and the drug-loaded nanoparticles with methotrexate. The methotrexate-loaded PLA-PEG-PLA nanoparticles have been studied in vitro; the drug release, drug loading, and yield are reported. Conclusion The drug release profile was monitored over a period of 168 hours, and was free of burst effect before the protein coating. The results obtained from this work are promising; this work can be taken further to develop MTX based therapies.

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures.

PubMed

Juhász, Tamás; Matta, Csaba; Somogyi, Csilla; Katona, Éva; Takács, Roland; Soha, Rudolf Ferenc; Szabó, István A; Cserháti, Csaba; Sződy, Róbert; Karácsonyi, Zoltán; Bakó, Eva; Gergely, Pál; Zákány, Róza

2014-03-01

Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6 days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05 Hz, 600 Pa; for 30 min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PKA) activity was also detected and CREB, the archetypal downstream transcription factor of PKA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PKA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesis-promoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of the intervention. Our results indicate that proper mechanical stimuli augment in vitro cartilage formation via promoting both differentiation and matrix production of chondrogenic cells, and the opposing regulation of the PKA/CREB-Sox9 and the PP2A signalling pathways is crucial in this phenomenon. Copyright © 2014 Elsevier Inc. All rights reserved.

Studies on improved integrated membrane-based chromatographic process for bioseparation

NASA Astrophysics Data System (ADS)

Xu, Yanke

To improve protein separation and purification directly from a fermentation broth, a novel membrane filtration-cum-chromatography device configuration having a relatively impermeable coated zone near the hollow fiber module outlet has been developed. The integrated membrane filtration-cum-chromatography unit packed with chromatographic beads on the shell side of the hollow fiber unit enjoys the advantages of both membrane filtration and chromatography; it allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane while leaving the rest of the hollow fiber membrane unaffected. Myoglobin (Mb), bovine serum albumin (BSA) and a-lactalbumin (a-LA) were used as model proteins in binary mixtures. Separation behaviors of binary protein mixtures were studied in devices using either an ultrafiltration (UF) membrane or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, the Mb and a-LA elution profiles for the four consecutive cyclic runs were almost superimposable. Due to the lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem as it is in conventional microfiltration. A mathematical model describing the hydrodynamic and protein loading behaviors of the integrated device using UF membrane with a coated zone was developed. The simulation results for the breakthrough agree well with the experimental breakthrough curves. The optimal length of the coated zone was obtained from the simulation. A theoretical analysis of the protein mass transfer was performed using a diffusion-convection model considering the feed-side concentration polarization and the permeate-side concentration gradient formed by the adsorption. The permeate-side adsorption can enhance the observed protein transmission through the membrane considerably at low permeate flux. But the enhancement effect can be neglected at higher permeate flux when convection dominates the total mass transfer process or the proteins are very highly rejected by the membrane.

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein.

PubMed

Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar; Kaur, Kamaljit; Suresh, Mavanur R

2012-04-02

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.

In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbon, Silvia, E-mail: silvia.barbon@yahoo.it

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in themore » future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT-CNTF was accomplished after cell treatment. • TAT-CNTF can be efficiently loaded on oxidized PVA cylinders for local delivery. • TAT-CNTF features make it ideal for peripheral nerve regeneration therapies.« less

Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response.

PubMed

Goldstein, Ido; Baek, Songjoon; Presman, Diego M; Paakinaho, Ville; Swinstead, Erin E; Hager, Gordon L

2017-03-01

Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting. Published by Cold Spring Harbor Laboratory Press.

Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model

PubMed Central

Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

2017-01-01

In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease. PMID:28186208

Whole eggs enhance antioxidant activity when combined with energy dense, cooked breakfast foods

USDA-ARS?s Scientific Manuscript database

Acute metabolic changes following the consumption of energy dense foods high in saturated fat (SFA) and glycemic load (GL) may contribute to the pathogenesis of several chronic diseases. Eggs provide highly digestible protein, unsaturated fatty acids, carotenoids, and other antioxidant compounds tha...

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway.

PubMed

Yan, Wenxia; Liu, Hanping; Deng, Xiaoyuan; Jin, Ying; Wang, Ning; Chu, Jing

2018-03-01

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin-induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha-smooth muscle actin and transforming growth factor-beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing. Copyright © 2017 John Wiley & Sons, Ltd.

Improved insulin loading in poly(lactic-co-glycolic) acid (PLGA) nanoparticles upon self-assembly with lipids.

PubMed

García-Díaz, María; Foged, Camilla; Nielsen, Hanne Mørck

2015-03-30

Polymeric nanoparticles are widely investigated as drug delivery systems for oral administration. However, the hydrophobic nature of many polymers hampers effective loading of the particles with hydrophilic macromolecules such as insulin. Thus, the aim of this work was to improve the loading of insulin into poly(lactic-co-glycolic) acid (PLGA) nanoparticles by pre-assembly with amphiphilic lipids. Insulin was complexed with soybean phosphatidylcholine or sodium caprate by self-assembly and subsequently loaded into PLGA nanoparticles by using the double emulsion-solvent evaporation technique. The nanoparticles were characterized in terms of size, zeta potential, insulin encapsulation efficiency and loading capacity. Upon pre-assembly with lipids, there was an increased distribution of insulin into the organic phase of the emulsion, eventually resulting in significantly enhanced encapsulation efficiencies (90% as compared to 24% in the absence of lipids). Importantly, the insulin loading capacity was increased up to 20% by using the lipid-insulin complexes. The results further showed that a main fraction of the lipid was incorporated into the nanoparticles and remained associated to the polymer during release studies in buffers, whereas insulin was released in a non-complexed form as a burst of approximately 80% of the loaded insulin. In conclusion, the protein load in PLGA nanoparticles can be significantly increased by employing self-assembled protein-lipid complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced oxidative stability of fish oil by encapsulating in culled banana resistant starch-soy protein isolate based microcapsules in functional bakery products.

PubMed

Nasrin, Taslima Ayesha Aktar; Anal, Anil Kumar

2015-08-01

Oil in water emulsions were produced by the mixture of culled banana resistant starch (CBRS) & soy protein isolate (SPI), mixture of Hylon VII & SPI and SPI with 7.5 and 5 % (w/w) Menhaden fish oil. The emulsions were further freeze- dried obtaining 33 and 50 % oil load microcapsules. The range of particles diameter was 4.11 to 7.25 μm and viscosity was 34.6 to 146.48 cP of the emulsions. Compressibility index (CI), Hasner ratio (HR) and angle of repose (AR) was significantly (p < 0.01) lower of the microcapsules made with starch and protein (CBRS & SPI and Hylon VII & SPI) than that made with protein (SPI) only. Microcapsules composed of CBRS & SPI with 33 % oil load had maximum microencapsulation efficiency (82.49 %) and highest oxidative stability. Muffin made with emulsions containing mixture of CBRS & SPI exhibited less fishy flavour than that containing mixture of Hylon VII & SPI.

Enhanced Sucrose Loading Improves Rice Yield by Increasing Grain Size1[OPEN

PubMed Central

Wang, Liang; Lu, Qingtao

2015-01-01

Yield in cereals is a function of grain number and size. Sucrose (Suc), the main carbohydrate product of photosynthesis in higher plants, is transported long distances from source leaves to sink organs such as seeds and roots. Here, we report that transgenic rice plants (Oryza sativa) expressing the Arabidopsis (Arabidopsis thaliana) phloem-specific Suc transporter (AtSUC2), which loads Suc into the phloem under control of the phloem protein2 promoter (pPP2), showed an increase in grain yield of up to 16% relative to wild-type plants in field trials. Compared with wild-type plants, pPP2::AtSUC2 plants had larger spikelet hulls and larger and heavier grains. Grain filling was accelerated in the transgenic plants, and more photoassimilate was transported from the leaves to the grain. In addition, microarray analyses revealed that carbohydrate, amino acid, and lipid metabolism was enhanced in the leaves and grain of pPP2::AtSUC2 plants. Thus, enhancing Suc loading represents a promising strategy to improve rice yield to feed the global population. PMID:26504138

TAF11 assembles RISC loading complex to enhance RNAi efficiency

PubMed Central

Liang, Chunyang; Wang, Yibing; Murota, Yukiko; Liu, Xiang; Smith, Dean; Siomi, Mikiko C.; Liu, Qinghua

2015-01-01

SUMMARY Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains Dicer-2(Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here, we identified the missing factor of RLC as TATA-binding protein associated factor 11 (TAF11) by genetic screen. Although an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11−/− ovary extract, we reconstituted the RLC in vitro using recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of Drosophila RLC and elucidate a novel cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency. PMID:26257286

Enhanced Sucrose Loading Improves Rice Yield by Increasing Grain Size.

PubMed

Wang, Liang; Lu, Qingtao; Wen, Xiaogang; Lu, Congming

2015-12-01

Yield in cereals is a function of grain number and size. Sucrose (Suc), the main carbohydrate product of photosynthesis in higher plants, is transported long distances from source leaves to sink organs such as seeds and roots. Here, we report that transgenic rice plants (Oryza sativa) expressing the Arabidopsis (Arabidopsis thaliana) phloem-specific Suc transporter (AtSUC2), which loads Suc into the phloem under control of the phloem protein2 promoter (pPP2), showed an increase in grain yield of up to 16% relative to wild-type plants in field trials. Compared with wild-type plants, pPP2::AtSUC2 plants had larger spikelet hulls and larger and heavier grains. Grain filling was accelerated in the transgenic plants, and more photoassimilate was transported from the leaves to the grain. In addition, microarray analyses revealed that carbohydrate, amino acid, and lipid metabolism was enhanced in the leaves and grain of pPP2::AtSUC2 plants. Thus, enhancing Suc loading represents a promising strategy to improve rice yield to feed the global population. © 2015 American Society of Plant Biologists. All Rights Reserved.

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes.

PubMed

Dutta, Shovan; Celestine, Michael J; Khanal, Supreet; Huddleston, Alexis; Simms, Colin; Arca, Jessa Faye; Mitra, Amlan; Heller, Loree; Kraj, Piotr J; Ledizet, Michel; Anderson, John F; Neelakanta, Girish; Holder, Alvin A; Sultana, Hameeda

2018-01-01

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100μM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen) 2 ]Cl 2 , (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen) 3 ]Cl 3 , (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl 2 ·2H 2 O) or cobalt(II) chloride hexahydrate (CoCl 2 ·6H 2 O) alone had no effects as "free" cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly. Copyright © 2017 Elsevier B.V. All rights reserved.

Minocycline modulates NFκB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

PubMed

Guerra, Alberto Daniel; Rose, Warren E; Hematti, Peiman; Kao, W John

2017-07-21

Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds. Modulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague-Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student's t test was used to determine p values for all assays with multiple or two conditions, respectively. Minocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH 2 -terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization. Minocycline modulates the NFκB pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.

Protein cage assisted metal-protein nanocomposite synthesis: Optimization of loading conditions

NASA Astrophysics Data System (ADS)

Sana, Barindra; Calista, Marcia; Lim, Sierin

2012-11-01

Ferritin is an iron-storage protein in most living systems with a cage-like structure. It has inherent property to form metallic nanocore within its cavity. The metallic core formed within the Archaeoglobus fulgidus ferritin cavity is stabilized by modulating the protein structure by site directed mutagenesis. Encapsulation protocol of various metals within the engineered ferritin cage (AfFtn-AA) is optimized. Dense metallic cores are visualized using electron microscopy and the bound metal was quantified by ICP-spectrometry. The AfFtn-AA is loaded with up to about 350 cobalt, 2000 chromium, and as high as 7000 iron atoms, separately. The metal-protein nanocomposites formed by encapsulation of cobalt, chromium, and iron are studied. Magnetic resonance imaging of the agarose embedded nanocomposites shows brightening of T1-weighted images and signal loss of T2-weighted images with increasing concentration of the nanocomposites. Shortening of magnetic relaxation times in the presence of the nanocomposites confirm their ability to enhance magnetic relaxation rate and suggests that the nanocomposites have potential application as MRI contrast agent.

Study of Mesoporous Silica Nanoparticles' (MSNs) intracellular trafficking and their application as drug delivery vehicles

NASA Astrophysics Data System (ADS)

Yanes, Rolando Eduardo

Mesoporous silica nanoparticles (MSNs) are attractive drug delivery vehicle candidates due to their biocompatibility, stability, high surface area and efficient cellular uptake. In this dissertation, I discuss three aspects of MSNs' cellular behavior. First, MSNs are targeted to primary and metastatic cancer cell lines, then their exocytosis from cancer cells is studied, and finally they are used to recover intracellular proteins. Targeting of MSNs to primary cancer cells is achieved by conjugating transferrin on the surface of the mesoporous framework, which resulted in enhancement of nanoparticle uptake and drug delivery efficacy in cells that overexpress the transferrin receptor. Similarly, RGD peptides are used to target metastatic cancer cell lines that over-express integrin alphanubeta3. A circular RGD peptide is bound to the surface of MSNs and the endocytosis and cell killing efficacy of camptothecin loaded nanoparticles is significantly improved in cells that express the target receptor. Besides targeting, I studied the ultimate fate of phosphonate coated mesoporous silica nanoparticles inside cells. I discovered that the nanoparticles are exocytosed from cells through lysosomal exocytosis. The nanoparticles are exocytosed in intact form and the time that they remain inside the cells is affected by the surface properties of the nanoparticles and the type of cells. Cells that have a high rate of lysosomal exocytosis excrete the nanoparticles rapidly, which makes them more resistant to drug loaded nanoparticles because the amount of drug that is released inside the cell is limited. When the exocytosis of MSNs is inhibited, the cell killing efficacy of nanoparticles loaded with camptothecin is enhanced. The discovery that MSNs are exocytosed by cells led to a study to determine if proteins could be recovered from the exocytosed nanoparticles. The procedure to isolate exocytosed zinc-doped iron core MSNs and identify the proteins bound to them was developed. This serves as a foundation to use MSNs as protein harvesting tools and investigate protein expression in cancer cells.

Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

PubMed Central

Chen, Shuang; Zhou, Liang; Zhang, Yu; Leng, Yun; Pei, Xin-Yan; Lin, Hui; Jones, Richard; Orlowski, Robert Z.

2014-01-01

In selective autophagy, the adaptor protein SQSTM1/p62 plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation. Here we report that whereas SQSTM1/p62 levels fluctuated in a time-dependent manner during autophagy, inhibition or knockdown of Cdk9/cyclin T1 transcriptionally downregulated SQSTM1/p62 but did not affect autophagic flux. These interventions, or short hairpin RNA (shRNA) directly targeting SQSTM1/p62, resulted in cargo loading failure and inefficient autophagy, phenomena recently described for Huntington's disease neurons. These events led to the accumulation of the BH3-only protein NBK/Bik on endoplasmic reticulum (ER) membranes, most likely by blocking loading and autophagic degradation of NBK/Bik, culminating in apoptosis. Whereas NBK/Bik upregulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine (CQ) or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., genetically by shRNA targeting Ulk1, beclin-1, or Atg5 or pharmacologically by 3-methyladenine [3-MA] or spautin-1), arguing that NBK/Bik accumulation stems from inefficient autophagy. Finally, NBK/Bik knockdown markedly attenuated apoptosis in vitro and in vivo. Together, these findings identify novel cross talk between autophagy and apoptosis, wherein targeting SQSTM1/p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation, which triggers apoptosis. PMID:25002530

Efficient antitumor effect of co-drug-loaded nanoparticles with gelatin hydrogel by local implantation

PubMed Central

Zhang, Hao; Tian, Yong; Zhu, Zhenshu; Xu, Huae; Li, Xiaolin; Zheng, Donghui; Sun, Weihao

2016-01-01

Tetrandrine (Tet) could enhance the antitumor effect of Paclitaxel (Ptx) by increasing intracellular Reactive Oxygen Species (ROS) levels, which leads to the possibility of co-delivery of both drugs for synergistic antitumor effect. In the current study, we reported an efficient, local therapeutic strategy employing effective Tet and Ptx delivery with a nanoparticle-loaded gelatin system. Tet- and Ptx co-loaded mPEG-PCL nanoparticles (P/T-NPs) were encapsulated into the physically cross-linked gelatin hydrogel and then implanted on the tumor site for continuous drug release. The drug-loaded gelatin hydrogel underwent a phase change when the temperature slowly increased. In vitro study showed that Tet/Ptx-loaded PEG-b-PCL nanoparticles encapsulated within a gelatin hydrogel (P/T-NPs-Gelatin) inhibited the growth and invasive ability of BGC-823 cells more effectively than the combination of free drugs or P/T-NPs. In vivo study validated the therapeutic potential of P/T-NPs-Gelatin. P/T-NPs-Gelatin significantly inhibited the activation of p-Akt and the downstream anti-apoptotic Bcl-2 protein and also inducing the activation of pro-apoptotic Bax protein. Moreover, the molecular-modulating effect of P/T-NPs-Gelatin on related proteins varied slightly under the influence of NAC, which was supported by the observations of the tumor volumes and weights. Based on these findings, local implantation of P/T-NPs-Gelatin may be a promising therapeutic strategy for the treatment of gastric cancer. PMID:27226240

Lycopene loaded whey protein isolate nanoparticles: An innovative endeavor for enhanced bioavailability of lycopene and anti-cancer activity.

PubMed

Jain, Ashay; Sharma, Gajanand; Ghoshal, Gargi; Kesharwani, Prashant; Singh, Bhupinder; Shivhare, U S; Katare, O P

2018-04-30

The work entails a novel strategy of formulating the lycopene loaded whey protein isolate nanoparticles (LYC-WPI-NPs) solely using the rational blend of biomacromolecule without using equipment-intensive techniques. The LYC-WPI-NPs were fabricated as a substantial drug delivery platform, with maximum entrapment, spatial and controlled release manners, exceptional plasma concentration, and perspective for discrepancy delivery of therapeutics. Prepared nano-formulations were measured in ultra-fine size (100-350 nm) with sphere-shaped. The percent lycopene entrapment of prepared LYC-WPI-NPs was estimated in the range to 50 and 65%. In vitro percent cumulative release study demonstrated deaden and extended release i.e. approximately 75% following 16th h. The in vitro percent cell survival (cytotoxicity study) of prepared nanoparticles was evaluated against MCF-7 breast cancer cells by MTT based colorimetric assay. Sub-cellular localization of lycopene when delivered by LYC-WPI-NPs was assessed by HPLC (high performance liquid chromatography). The WPI-NPs enhance the oral bioavailability of lycopene by controlling its release from nano-formulation and facilitating its absorption through lymphatic pathways. Prophylactic anticancer efficacy of LYC-WPI-NPs was evaluated thereafter on experimentally induced breast cancer animal model. Conclusively, it may quite reasonable that lycopene loaded protein nanoparticles are competent to improve the biopharmaceutical attributes of lycopene and demonstrated prophylactic anticancer activity, decrease tumor proliferation and increase the survival rate of treated animals, thus signifying their feasible usefulness in cancer therapeutic and intervention. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel aptamer-nanoparticle bioconjugates enhances delivery of anticancer drug to MUC1-positive cancer cells in vitro.

PubMed

Yu, Chenchen; Hu, Yan; Duan, Jinhong; Yuan, Wei; Wang, Chen; Xu, Haiyan; Yang, Xian-Da

2011-01-01

MUC1 protein is an attractive target for anticancer drug delivery owing to its overexpression in most adenocarcinomas. In this study, a reported MUC1 protein aptamer is exploited as the targeting agent of a nanoparticle-based drug delivery system. Paclitaxel (PTX) loaded poly (lactic-co-glycolic-acid) (PLGA) nanoparticles were formulated by an emulsion/evaporation method, and MUC1 aptamers (Apt) were conjugated to the particle surface through a DNA spacer. The aptamer conjugated nanoparticles (Apt-NPs) are about 225.3 nm in size with a stable in vitro drug release profile. Using MCF-7 breast cancer cell as a MUC1-overexpressing model, the MUC1 aptamer increased the uptake of nanoparticles into the target cells as measured by flow cytometry. Moreover, the PTX loaded Apt-NPs enhanced in vitro drug delivery and cytotoxicity to MUC1(+) cancer cells, as compared with non-targeted nanoparticles that lack the MUC1 aptamer (P<0.01). The behavior of this novel aptamer-nanoparticle bioconjugates suggests that MUC1 aptamers may have application potential in targeted drug delivery towards MUC1-overexpressing tumors.

Emodin self-emulsifying platform ameliorates the expression of FN, ICAM-1 and TGF-β1 in AGEs-induced glomerular mesangial cells by promoting absorption.

PubMed

Huang, Jiani; Gong, Wenyan; Chen, Zhiquan; Huang, Junying; Chen, Qiuhong; Huang, Heqing; Zhao, Chunshun

2017-03-01

Emodin, a potential anti-diabetic nephropathy agent, is limited by its oral use due to the poor water solubility. The present study aimed to enhance the absorption and the suppressive effects of emodin on renal fibrosis by developing a self-microemulsifying drug delivery system (SMEDDS). Solubility studies, compatibility tests, pseudo-ternary phase diagrams analysis and central composite design were carried out to obtain the optimized formulation. The average droplet size of emodin-loaded SMEDDS was about 18.31±0.12nm, and the droplet size and zeta potential remained stable at different dilution ratios of water and different values of pH varying from 1.2 to 7.2. Enhanced cellular uptake in both the Caco-2 cells and glomerular mesangial cells (GMCs) is great advantageous for the formulation. The AUC 0-24h of emodin-loaded SMEDDS was 1.87-fold greater than that of emodin suspension, which may be attributed to enhanced uptake in Caco-2 cells. Moreover, emodin-loaded SMEDDS showed better suppressive effects on the protein level of fibronectin (FN), transforming growth factor-beta 1 (TGF-β1) and intercellular adhesion molecule 1 (ICAM-1) than the crude emodin in advanced glycation-end products (AGEs)-induced GMCs and renal tubular epithelial cells (NRK-52E). Our study illustrated that developed SMEDDS improved the oral absorption of emodin, and attained better suppressive effects on the protein level of renal fibrosis compositions in AGEs-induced GMCs and NRK-52E cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response

PubMed Central

Canova, Donata Federici; Pavlov, Anton M.; Norling, Lucy V.; Gobbetti, Thomas; Brunelleschi, Sandra; Le Fauder, Pauline; Cenac, Nicolas; Sukhorukov, Gleb B.; Perretti, Mauro

2015-01-01

Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis. PMID:26385167

The acute angiogenic signalling response to low-load resistance exercise with blood flow restriction.

PubMed

Ferguson, Richard A; Hunt, Julie E A; Lewis, Mark P; Martin, Neil R W; Player, Darren J; Stangier, Carolin; Taylor, Conor W; Turner, Mark C

2018-04-01

This study investigated protein kinase activation and gene expression of angiogenic factors in response to low-load resistance exercise with or without blood flow restriction (BFR). In a repeated measures cross-over design, six males performed four sets of bilateral knee extension exercise at 20% 1RM (reps per set = 30:15:15:continued to fatigue) with BFR (110 mmHg) and without (CON). Muscle biopsies were obtained from the vastus lateralis before, 2 and 4 h post-exercise. mRNA expression was determined using real-time RT-PCR. Protein phosphorylation/expression was determined using Western blot. p38MAPK phosphorylation was greater (p = 0.05) at 2 h following BFR (1.3 ± 0.8) compared to CON (0.4 ± 0.3). AMPK phosphorylation remained unchanged. PGC-1α mRNA expression increased at 2 h (5.9 ± 1.3 vs. 2.1 ± 0.8; p = 0.03) and 4 h (3.2 ± 0.8 vs. 1.5 ± 0.4; p = 0.03) following BFR exercise with no change in CON. PGC-1α protein expression did not change following either exercise. BFR exercise enhanced mRNA expression of vascular endothelial growth factor (VEGF) at 2 h (5.2 ± 2.8 vs 1.7 ± 1.1; p = .02) and 4 h (6.8 ± 4.9 vs. 2.5 ± 2.7; p = .01) compared to CON. mRNA expression of VEGF-R2 and hypoxia-inducible factor 1α increased following BFR exercise but only eNOS were enhanced relative to CON. Matrix metalloproteinase-9 mRNA expression was not altered in response to either exercise. Acute low-load resistance exercise with BFR provides a targeted angiogenic response potentially mediated through enhanced ischaemic and shear stress stimuli.

TAF11 Assembles the RISC Loading Complex to Enhance RNAi Efficiency.

PubMed

Liang, Chunyang; Wang, Yibing; Murota, Yukiko; Liu, Xiang; Smith, Dean; Siomi, Mikiko C; Liu, Qinghua

2015-09-03

Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here we identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11(-/-) ovary extract, we reconstituted the RLC in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency. Copyright © 2015 Elsevier Inc. All rights reserved.

Human Muscle Protein Synthetic Responses during Weight-Bearing and Non-Weight-Bearing Exercise: A Comparative Study of Exercise Modes and Recovery Nutrition.

PubMed

Pasiakos, Stefan M; McClung, Holly L; Margolis, Lee M; Murphy, Nancy E; Lin, Gregory G; Hydren, Jay R; Young, Andrew J

2015-01-01

Effects of conventional endurance (CE) exercise and essential amino acid (EAA) supplementation on protein turnover are well described. Protein turnover responses to weighted endurance exercise (i.e., load carriage, LC) and EAA may differ from CE, because the mechanical forces and contractile properties of LC and CE likely differ. This study examined muscle protein synthesis (MPS) and whole-body protein turnover in response to LC and CE, with and without EAA supplementation, using stable isotope amino acid tracer infusions. Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, VO 2peak 4.0 ± 0.5 L ∙ min(-1)) were randomly assigned to perform 90 min, absolute intensity-matched (2.2 ± 0.1 VO2 L ∙ m(-1)) LC (performed on a treadmill wearing a vest equal to 30% of individual body mass, mean ± SD load carried 24 ± 3 kg) or CE (cycle ergometry performed at the same absolute VO2 as LC) exercise, during which EAA (10 g EAA, 3.6 g leucine) or control (CON, non-nutritive) drinks were consumed. Mixed-muscle and myofibrillar MPS were higher during exercise for LC than CE (mode main effect, P < 0.05), independent of dietary treatment. EAA enhanced mixed-muscle and sarcoplasmic MPS during exercise, regardless of mode (drink main effect, P < 0.05). Mixed-muscle and sarcoplasmic MPS were higher in recovery for LC than CE (mode main effect, P < 0.05). No other differences or interactions (mode x drink) were observed. However, EAA attenuated whole-body protein breakdown, increased amino acid oxidation, and enhanced net protein balance in recovery compared to CON, regardless of exercise mode (P < 0.05). These data show that, although whole-body protein turnover responses to absolute VO2-matched LC and CE are the same, LC elicited a greater muscle protein synthetic response than CE.

Peptide/protein vaccine delivery system based on PLGA particles.

PubMed

Allahyari, Mojgan; Mohit, Elham

2016-03-03

Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted.

Peptide/protein vaccine delivery system based on PLGA particles

PubMed Central

Allahyari, Mojgan; Mohit, Elham

2016-01-01

abstract Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted. PMID:26513024

A thiol probe for measuring unfolded protein load and proteostasis in cells.

PubMed

Chen, Moore Z; Moily, Nagaraj S; Bridgford, Jessica L; Wood, Rebecca J; Radwan, Mona; Smith, Trevor A; Song, Zhegang; Tang, Ben Zhong; Tilley, Leann; Xu, Xiaohong; Reid, Gavin E; Pouladi, Mahmoud A; Hong, Yuning; Hatters, Danny M

2017-09-07

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

In vitro study of novel gadolinium-loaded liposomes guided by GBI-10 aptamer for promising tumor targeting and tumor diagnosis by magnetic resonance imaging.

PubMed

Gu, Meng-Jie; Li, Kun-Feng; Zhang, Lan-Xin; Wang, Huan; Liu, Li-Si; Zheng, Zhuo-Zhao; Han, Nan-Yin; Yang, Zhen-Jun; Fan, Tian-Yuan

2015-01-01

Novel gadolinium-loaded liposomes guided by GBI-10 aptamer were developed and evaluated in vitro to enhance magnetic resonance imaging (MRI) diagnosis of tumor. Nontargeted gadolinium-loaded liposomes were achieved by incorporating amphipathic material, Gd (III) [N,N-bis-stearylamidomethyl-N'-amidomethyl] diethylenetriamine tetraacetic acid, into the liposome membrane using lipid film hydration method. GBI-10, as the targeting ligand, was then conjugated onto the liposome surface to get GBI-10-targeted gadolinium-loaded liposomes (GTLs). Both nontargeted gadolinium-loaded liposomes and GTLs displayed good dispersion stability, optimal size, and zeta potential for tumor targeting, as well as favorable imaging properties with enhanced relaxivity compared with a commercial MRI contrast agent (CA), gadopentetate dimeglumine. The use of GBI-10 aptamer in this liposomal system was intended to result in increased accumulation of gadolinium at the periphery of C6 glioma cells, where the targeting extracellular matrix protein tenascin-C is overexpressed. Increased cellular binding of GTLs to C6 cells was confirmed by confocal microscopy, flow cytometry, and MRI, demonstrating the promise of this novel delivery system as a carrier of MRI contrast agent for the diagnosis of tumor. These studies provide a new strategy furthering the development of nanomedicine for both diagnosis and therapy of tumor.

2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity.

PubMed

Wu, Sherry Y; Yang, Xianbin; Gharpure, Kshipra M; Hatakeyama, Hiroto; Egli, Martin; McGuire, Michael H; Nagaraja, Archana S; Miyake, Takahito M; Rupaimoole, Rajesha; Pecot, Chad V; Taylor, Morgan; Pradeep, Sunila; Sierant, Malgorzata; Rodriguez-Aguayo, Cristian; Choi, Hyun J; Previs, Rebecca A; Armaiz-Pena, Guillermo N; Huang, Li; Martinez, Carlos; Hassell, Tom; Ivan, Cristina; Sehgal, Vasudha; Singhania, Richa; Han, Hee-Dong; Su, Chang; Kim, Ji Hoon; Dalton, Heather J; Kovvali, Chandra; Keyomarsi, Khandan; McMillan, Nigel A J; Overwijk, Willem W; Liu, Jinsong; Lee, Ju-Seog; Baggerly, Keith A; Lopez-Berestein, Gabriel; Ram, Prahlad T; Nawrot, Barbara; Sood, Anil K

2014-03-12

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2'-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.

The effect of caffeine on skeletal muscle anabolic signaling and hypertrophy.

PubMed

Moore, Timothy M; Mortensen, Xavier M; Ashby, Conrad K; Harris, Alexander M; Kump, Karson J; Laird, David W; Adams, Aaron J; Bray, Jeremy K; Chen, Ting; Thomson, David M

2017-06-01

Caffeine is a widely consumed stimulant with the potential to enhance physical performance through multiple mechanisms. However, recent in vitro findings have suggested that caffeine may block skeletal muscle anabolic signaling through AMP-activated protein kinase (AMPK)-mediated inhibition of mechanistic target of rapamycin (mTOR) signaling pathway. This could negatively affect protein synthesis and the capacity for muscle growth. The primary purpose of this study was to assess the effect of caffeine on in vivo AMPK and mTOR pathway signaling, protein synthesis, and muscle growth. In cultured C2C12 muscle cells, physiological levels of caffeine failed to impact mTOR activation or myoblast proliferation or differentiation. We found that caffeine administration to mice did not significantly enhance the phosphorylation of AMPK or inhibit signaling proteins downstream of mTOR (p70S6k, S6, or 4EBP1) or protein synthesis after a bout of electrically stimulated contractions. Skeletal muscle-specific knockout of LKB1, the primary AMPK activator in skeletal muscle, on the other hand, eliminated AMPK activation by contractions and enhanced S6k, S6, and 4EBP1 activation before and after contractions. In rats, the addition of caffeine did not affect plantaris hypertrophy induced by the tenotomy of the gastrocnemius and soleus muscles. In conclusion, caffeine administration does not impair skeletal muscle load-induced mTOR signaling, protein synthesis, or muscle hypertrophy.

A protein/antibiotic releasing poly(lactic-co-glycolic acid)/lecithin scaffold for bone repair applications.

PubMed

Shi, Xuetao; Wang, Yingjun; Ren, Li; Huang, Wei; Wang, Dong-An

2009-05-21

Novel poly(lactic-co-glycolic acid) (PLGA)-hybridizing-lecithin scaffolds loaded with drug or protein were prepared with water/oil/water techniques and sintering microspheres technique. In such fabricated composite scaffolds (abbreviated "PLGA/Lec-SMS"), the introduction of lecithin component has been proven capable of largely enhancing Gentamicin (GS) and protein (Bovine Serum Albumin) encapsulation efficiency. The in vitro GS and BSA releasing profiles of PLGA/Lec-SMS system were plotted basing over 60 days' and 18 days' data collection, respectively. It indicates a sustained releasing tendency despite a burst at the very beginning. The antibacterial properties of GS-laden scaffolds were determined in vitro, and the antibacterial activity of scaffolds was enhanced by incorporating lecithin into PLGA bulks. Additionally, mesenchymal stem cells (MSCs) were seeded onto PLGA-SMS and PLGA/Lec-SMS in vitro. The outcome confirmed PLGA/Lec(5%)-SMS functions to improve MSC proliferation and also to enhance general ALP production and calcium secretion which is the vital markers for osteogenesis. In conclusion, this newly designed antibiotic releasing PLGA/Lec-SMS is promising for bone-repairing therapeutics.

Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Mertz, Damien; Affolter-Zbaraszczuk, Christine; Barthès, Julien; Cui, Jiwei; Caruso, Frank; Baumert, Thomas F.; Voegel, Jean-Claude; Ogier, Joelle; Meyer, Florent

2014-09-01

In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing.In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing. Electronic supplementary information (ESI) available: Experimental details and supporting Fig. S1-S4. See DOI: 10.1039/c4nr02623c

Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading

NASA Technical Reports Server (NTRS)

Decker, R. S.; Cook, M. G.; Behnke-Barclay, M.; Decker, M. L.

1995-01-01

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Real‐time monitoring and control of the load phase of a protein A capture step

PubMed Central

Rüdt, Matthias; Brestrich, Nina; Rolinger, Laura

2016-01-01

ABSTRACT The load phase in preparative Protein A capture steps is commonly not controlled in real‐time. The load volume is generally based on an offline quantification of the monoclonal antibody (mAb) prior to loading and on a conservative column capacity determined by resin‐life time studies. While this results in a reduced productivity in batch mode, the bottleneck of suitable real‐time analytics has to be overcome in order to enable continuous mAb purification. In this study, Partial Least Squares Regression (PLS) modeling on UV/Vis absorption spectra was applied to quantify mAb in the effluent of a Protein A capture step during the load phase. A PLS model based on several breakthrough curves with variable mAb titers in the HCCF was successfully calibrated. The PLS model predicted the mAb concentrations in the effluent of a validation experiment with a root mean square error (RMSE) of 0.06 mg/mL. The information was applied to automatically terminate the load phase, when a product breakthrough of 1.5 mg/mL was reached. In a second part of the study, the sensitivity of the method was further increased by only considering small mAb concentrations in the calibration and by subtracting an impurity background signal. The resulting PLS model exhibited a RMSE of prediction of 0.01 mg/mL and was successfully applied to terminate the load phase, when a product breakthrough of 0.15 mg/mL was achieved. The proposed method has hence potential for the real‐time monitoring and control of capture steps at large scale production. This might enhance the resin capacity utilization, eliminate time‐consuming offline analytics, and contribute to the realization of continuous processing. Biotechnol. Bioeng. 2017;114: 368–373. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc. PMID:27543789

A polymeric fastener can easily functionalize liposome surfaces with gadolinium for enhanced magnetic resonance imaging.

PubMed

Smith, Cartney E; Shkumatov, Artem; Withers, Sarah G; Yang, Binxia; Glockner, James F; Misra, Sanjay; Roy, Edward J; Wong, Chun-Ho; Zimmerman, Steven C; Kong, Hyunjoon

2013-11-26

Common methods of loading magnetic resonance imaging (MRI) contrast agents into nanoparticles often suffer from challenges related to particle formation, complex chemical modification/purification steps, and reduced contrast efficiency. This study presents a simple, yet advanced process to address these issues by loading gadolinium, an MRI contrast agent, exclusively on a liposome surface using a polymeric fastener. The fastener, so named for its ability to physically link the two functional components together, consisted of chitosan substituted with diethylenetriaminepentaacetic acid (DTPA) to chelate gadolinium, as well as octadecyl chains to stabilize the modified chitosan on the liposome surface. The assembly strategy, mimicking the mechanisms by which viruses and proteins naturally anchor to a cell, provided greater T1 relaxivity than liposomes loaded with gadolinium in both the interior and outer leaflet. Gadolinium-coated liposomes were ultimately evaluated in vivo using murine ischemia models to highlight the diagnostic capability of the system. Taken together, this process decouples particle assembly and functionalization and, therefore, has considerable potential to enhance imaging quality while alleviating many of the difficulties associated with multifunctional particle fabrication.

A Polymeric Fastener can Easily Functionalize Liposome Surfaces with Gadolinium for Enhanced Magnetic Resonance Imaging

PubMed Central

Smith, Cartney E.; Shkumatov, Artem; Withers, Sarah G.; Glockner, James F.; Misra, Sanjay; Roy, Edward J.; Wong, Chun-Ho; Zimmerman, Steven C.; Kong, Hyunjoon

2013-01-01

Common methods of loading magnetic resonance imaging (MRI) contrast agents into nanoparticles often suffer from challenges related to particle formation, complex chemical modification/purification steps, and reduced contrast efficiency. This study presents a simple, yet advanced process to address these issues by loading gadolinium, an MRI contrast agent, exclusively on a liposome surface using a polymeric fastener. The fastener, so named for its ability to physically link the two functional components together, consisted of chitosan substituted with diethylenetriaminepentaacetic acid (DTPA) to chelate gadolinium, as well as octadecyl chains to stabilize the modified chitosan on the liposome surface. The assembly strategy, mimicking the mechanisms by which viruses and proteins naturally anchor to a cell, provided greater T1 relaxivity than liposomes loaded with gadolinium in both the interior and outer leaflet. Gadolinium-coated liposomes were ultimately evaluated in vivo using murine ischemia models to highlight the diagnostic capability of the system. Taken together, this process decouples particle assembly and functionalization, and therefore has considerable potential to enhance imaging quality while alleviating many of the difficulties associated with multifunctional particle fabrication. PMID:24083377

Low-magnitude mechanical vibration regulates expression of osteogenic proteins in ovariectomized rats.

PubMed

Li, Ming; Wu, Wei; Tan, Lei; Mu, Degong; Zhu, Dong; Wang, Jian; Zhao, Bin

2015-09-25

The present study aimed to investigate the impact of low-magnitude and high-frequency mechanical vibration with various lengths of resting period incorporated between loading cycles on the expression of osteogenesis-related proteins in a rat model of osteoporosis. The rats in the mechanical loading groups received low-magnitude and high-frequency vibration (35 Hz and acceleration of 0.25 g, 15 min/day) for 8 weeks. Bilateral humeral heads and femoral heads were then isolated, and protein levels of bone morphogenetic protein 2 (BMP-2), extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were determined by Western blotting. Increased levels of BMP-2, Runx2 and OCN were observed in rats receiving mechanical vibration. Total ERK1/2 protein remained unchanged, whereas the level of activated ERK1/2 (p-ERK1/2) increased after mechanical vibration. Vibration with incorporated resting period, regardless of length, was more effective in inducing expression of these osteogenic proteins, and the vibration with 7-day resting period had the most profound impact. Signals from low-magnitude and high-frequency mechanical vibration upregulated the expression of BMP-2 and Runx2, activated the ERK1/2 signaling pathway, and consequently led to increased expression of OCN. The anabolic effect of mechanical stimulation was enhanced with incorporation of resting period between loadings, and the one with 7-day resting period exhibited the strongest effect among all. Our results could provide a reference for development of mechanical stimulation as a non-pharmacological intervention for osteoporosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Phelligridin D-loaded oral nanotube titanium implant enhances osseointegration and prevents osteolysis in rat mandible.

PubMed

Kim, Ji-Eun; Takanche, Jyoti Shrestha; Kim, Jeong-Seok; Lee, Min-Ho; Jeon, Jae-Gyu; Park, Il-Song; Yi, Ho-Keun

2018-04-12

Poor bone quality and osteolysis are the major causes of implant failure in dentistry. Here, this study tested the effect of phelligridin D-loaded nanotubes titanium (Ti) for bone formation around the dental implants. The purpose of this study was to enhance osseointegration of phelligridin D-loaded implant into the bone for bone formation and prevention of osteolysis. Cell viability, crystal violet staining, Western blot, alizarin red S staining, alkaline phosphatase activity, tartrate-resistant acid phosphatase staining, micro-computed tromography (μ-CT), hematoxylin and eosin (H&E) and immunohistochemical staining were used in vitro and in vivo to test the biocompatibility of phelligridin D. Phelligridin D enhanced osteoblast differentiation and mineralization by increasing bone morphogenic protein-2/7 (BMP-2/7), Osterix, Runx-2, osteoprotegerin (OPG), alkaline phosphatase and inhibited osteoclast differentiation by decreasing receptor activator of nuclear factor kappa-B ligand (RANKL) in MC-3T3 E1 cells. Further, phelligridin D promoted bone regeneration around nanotube Ti implant surface by increasing the levels of BMP-2/7 and OPG in a rat model. Phelligridin D also inhibited osteolysis by suppressing the expression of RANKL. These findings strongly suggest that phelligridin D is a new compound representing a potential therapeutic candidate for implant failure caused by osteolysis and poor bone quality of teeth.

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

PubMed

Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.

Protein Nanoscaffolds for Delivering Toxic Inorganic Cargo to Cancer Cells

NASA Astrophysics Data System (ADS)

Cioloboc, Daniela

Targeted delivery of anticancer drugs or prodrugs to tumors can minimize systemic toxicity and side effects. This study develops platforms for targeted delivery of two potentially less systemically toxic prodrugs by exploiting the native and/or bioinorganic properties of two ferritins, both of which function naturally as iron storage proteins. Two delivery approaches were investigated. The first system was designed to serve as either an enhancement or alternative to traditional photodynamic therapy by generating hydroxyl radical in addition to singlet oxygen as the toxic reactive oxygen species. This system used Escherichia coli bacterioferritin (Bfr) loaded with 2,500 irons and multiple zinc-porphyrin (ZnP) photosensitizers. Ferrous iron was released by photoreduction of ferric iron stored within the Bfr protein shell. Hydroxyl radicals were generated via the Fenton reaction between hydrogen peroxide and the released ferrous iron. The outer surface of the Bfr protein shell was coated with peptides that specifically bind to a receptor known to be overexpressed in many tumor cells and tumor vasculature. The iron-loaded peptide-ZnP-Bfr was endocytosed by melanoma cells, where it showed photo-triggered release of iron and light-dependent cytotoxicity. The second system, built around human heavy chain ferritin (HFn), was loaded with arsenate as a less toxic "prodrug" and designed to release arsenic in its toxic, therapeutically effective reduced form, arsenic trioxide (ATO). The Hfn shell was coated with peptides targeting receptors that are hyperexpressed in triple negative breast cancers. The arsenate/iron-loaded-Hfn was endocytosed by a breast cancer cell line and showed cytotoxicity equivalent to that of free ATO on an arsenic basis, whereas the "empty" or iron-only loaded Hfn showed no cytotoxicity. Although HFn has previously been used to deliver organic drugs and imaging agents, these new results demonstrate that both Bfr and HFn can be manipulated to function as 'Trojan horse' nanocarriers for inorganic drugs.

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems

PubMed Central

Gand, Adeline; Hindié, Mathilde; Chacon, Diane; van Tassel, Paul R; Pauthe, Emmanuel

2014-01-01

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface). PMID:25482416

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems. Application to the growth factor BMP-2.

PubMed

Gand, Adeline; Hindié, Mathilde; Chacon, Diane; Van Tassel, Paul R; Pauthe, Emmanuel

2014-01-01

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface).

In vitro study of novel gadolinium-loaded liposomes guided by GBI-10 aptamer for promising tumor targeting and tumor diagnosis by magnetic resonance imaging

PubMed Central

Gu, Meng-Jie; Li, Kun-Feng; Zhang, Lan-Xin; Wang, Huan; Liu, Li-Si; Zheng, Zhuo-Zhao; Han, Nan-Yin; Yang, Zhen-Jun; Fan, Tian-Yuan

2015-01-01

Novel gadolinium-loaded liposomes guided by GBI-10 aptamer were developed and evaluated in vitro to enhance magnetic resonance imaging (MRI) diagnosis of tumor. Nontargeted gadolinium-loaded liposomes were achieved by incorporating amphipathic material, Gd (III) [N,N-bis-stearylamidomethyl-N′-amidomethyl] diethylenetriamine tetraacetic acid, into the liposome membrane using lipid film hydration method. GBI-10, as the targeting ligand, was then conjugated onto the liposome surface to get GBI-10-targeted gadolinium-loaded liposomes (GTLs). Both nontargeted gadolinium-loaded liposomes and GTLs displayed good dispersion stability, optimal size, and zeta potential for tumor targeting, as well as favorable imaging properties with enhanced relaxivity compared with a commercial MRI contrast agent (CA), gadopentetate dimeglumine. The use of GBI-10 aptamer in this liposomal system was intended to result in increased accumulation of gadolinium at the periphery of C6 glioma cells, where the targeting extracellular matrix protein tenascin-C is overexpressed. Increased cellular binding of GTLs to C6 cells was confirmed by confocal microscopy, flow cytometry, and MRI, demonstrating the promise of this novel delivery system as a carrier of MRI contrast agent for the diagnosis of tumor. These studies provide a new strategy furthering the development of nanomedicine for both diagnosis and therapy of tumor. PMID:26316749

Adaptation to high throughput batch chromatography enhances multivariate screening.

PubMed

Barker, Gregory A; Calzada, Joseph; Herzer, Sibylle; Rieble, Siegfried

2015-09-01

High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity.

PubMed

Li, Kai; Wang, Jianhua; He, Yaojia; Abdulrazaq, Miaad Adnan; Yan, Yunjun

2018-06-19

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu 3 (PO 4 ) 2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu 3 (PO 4 ) 2 /CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu 3 (PO 4 ) 2 /Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ee s ) reached 98% in only 10 min, and the corresponding Cu 3 (PO 4 ) 2 /CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application. Copyright © 2018 Elsevier B.V. All rights reserved.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

PubMed Central

MacAlpine, Heather K.; Lubelsky, Yoav; Hartemink, Alexander J.

2015-01-01

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2–7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we “footprinted” nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2–7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). PMID:25593310

The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

PubMed Central

You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

2015-01-01

ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading. PMID:26092121

Biomimetic chimeric peptide-tethered hydrogels for human mesenchymal stem cell delivery.

PubMed

Shim, Gayong; Kim, Gunwoo; Choi, Junhyeok; Yi, TacGhee; Cho, Yun Kyoung; Song, Sun Uk; Byun, Youngro; Oh, Yu-Kyoung

2015-12-01

Here, we report a chimeric peptide-tethered fibrin hydrogel scaffold for delivery of human mesenchymal stem cells (hMSC). Osteopontin-derived peptide (OP) was used as an hMSC-tethering moiety. OP showed hMSC adhesion properties and enhanced hMSC proliferation. A natural fibrin-binding protein-derived peptide (FBP) was tested for its ability to tether hMSC to the fibrin gel matrix. FBP loading on fibrin gels was 8.2-fold higher than that of a scrambled peptide (scFBP). FBP-loaded fibrin gels were retained at injection sites longer than scFBP-loaded fibrin gels, showing a 15.9-fold higher photon intensity of fluorescent FBP-grafted fibrin gels than fluorescent scFBP-loaded fibrin gels 48 h after injection. On the basis of the fibrin gel-binding properties of FBP and the hMSC-binding and proliferation-supporting properties of OP, we constructed chimeric peptides containing FBP and OP linked with a spacer (FBPsOP). Four days after transplantation, the survival of hMSC in FBPsOP-grafted fibrin gels was 3.9-fold higher than hMSC in fibrin gels alone. Our results suggest the potential of FBPsOP-grafted fibrin gels as a bioactive delivery system for enhanced survival of stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhancement of physical stability and bioaccessibility of tangeretin by soy protein isolate addition.

PubMed

Wan, Jiawei; Li, Dong; Song, Rong; Shah, Bakht Ramin; Li, Bin; Li, Yan

2017-04-15

Soy protein isolate (SPI) was selected to fabricate supersaturated self-emulsifying nanoemulsions, aiming to enhance physical stability and bioaccessibility of hydrophobic tangeretin. Dissolution studies demonstrated that tangeretin had the highest solubility in Tween 80, followed by oil phase solutions, and polymer solutions. Supersaturated tangeretin in oil phases easily formed crystals. That metastable zone was found to vary with its initial concentrations. After encapsulation by nanoemulsions, the addition of glycerol compressed the retention amount of tangeretin from 76% to 53%, but benefited the transparency. Whereas, the combination of glycerol and SPI could not only maintain high-loading tangeretin (>85%), but also provide high transparency for nanoemulsions. When tangeretin concentration was 4.83mM, combination of 50% glycerol and 1% SPI could maintain around 88% tangeretin in the nanoemulsion within one month. Its bioaccessibility of different systems were at 60-65%. These findings can provide useful information for protein to be a potential precipitation inhibitor. Copyright © 2016 Elsevier Ltd. All rights reserved.

Does a high dietary acid content cause bone loss, and can bone loss be prevented with an alkaline diet?

PubMed

Hanley, David A; Whiting, Susan J

2013-01-01

A popular concept in nutrition and lay literature is that of the role of a diet high in acid or protein in the pathogenesis of osteoporosis. A diet rich in fruit and vegetable intake is thought to enhance bone health as the result of its greater potassium and lower "acidic" content than a diet rich in animal protein and sodium. Consequently, there have been a number of studies of diet manipulation to enhance potassium and "alkaline" content of the diet to improve bone density or other parameters of bone health. Although acid loading or an acidic diet featuring a high protein intake may be associated with an increase in calciuria, the evidence supporting a role of these variables in the development of osteoporosis is not consistent. Similarly, intervention studies with a more alkaline diet or use of supplements of potassium citrate or bicarbonate have not consistently shown a bone health benefit. In the elderly, inadequate protein intake is a greater problem for bone health than protein excess. Copyright © 2013 The International Society for Clinical Densitometry. Published by Elsevier Inc. All rights reserved.

2’f-OMe-phosphorodithioate modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity

PubMed Central

Wu, Sherry Y.; Yang, Xianbin; Gharpure, Kshipra M.; Hatakeyama, Hiroto; Egli, Martin; McGuire, Michael H.; Nagaraja, Archana S.; Miyake, Takahito M.; Rupaimoole, Rajesha; Pecot, Chad V.; Taylor, Morgan; Pradeep, Sunila; Sierant, Malgorzata; Rodriguez-Aguayo, Cristian; Choi, Hyun J.; Previs, Rebecca A.; Armaiz-Pena, Guillermo N.; Huang, Li; Martinez, Carlos; Hassell, Tom; Ivan, Cristina; Sehgal, Vasudha; Singhania, Richa; Han, Hee-Dong; Su, Chang; Kim, Ji Hoon; Dalton, Heather J.; Kowali, Chandra; Keyomarsi, Khandan; McMillan, Nigel A.J.; Overwijk, Willem W.; Liu, Jinsong; Lee, Ju-Seog; Baggerly, Keith A.; Lopez-Berestein, Gabriel; Ram, Prahlad T.; Nawrot, Barbara; Sood, Anil K.

2014-01-01

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2’-O-Methyl (2’-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2’-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM Domain Containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumors following MePS2-modified siRNA treatment, leading to a synergistic anti-tumor effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types. PMID:24619206

Enhanced intracellular delivery and antibacterial efficacy of enrofloxacin-loaded docosanoic acid solid lipid nanoparticles against intracellular Salmonella.

PubMed

Xie, Shuyu; Yang, Fei; Tao, Yanfei; Chen, Dongmei; Qu, Wei; Huang, Lingli; Liu, Zhenli; Pan, Yuanhu; Yuan, Zonghui

2017-01-23

Enrofloxacin-loaded docosanoic acid solid lipid nanoparticles (SLNs) with different physicochemical properties were developed to enhance activity against intracellular Salmonella. Their cellular uptake, intracellular elimination and antibacterial activity were studied in RAW 264.7 cells. During the experimental period, SLN-encapsulated enrofloxacin accumulated in the cells approximately 27.06-37.71 times more efficiently than free drugs at the same extracellular concentration. After incubation for 0.5 h, the intracellular enrofloxacin was enhanced from 0.336 to 1.147 μg/mg of protein as the sizes of nanoparticles were increased from 150 to 605 nm, and from 0.960 to 1.147 μg/mg of protein when the charge was improved from -8.1 to -24.9 mv. The cellular uptake was more significantly influenced by the size than it was by the charge, and was not affected by whether the charge was positive or negative. The elimination of optimal SLN-encapsulated enrofloxacin from the cells was significantly slower than that of free enrofloxacin after removing extracellular drug. The inhibition effect against intracellular Salmonella CVCC541 of 0.24 and 0.06 μg/mL encapsulated enrofloxacin was stronger than 0.6 μg/mL free drug after all of the incubation periods and at 48 h, respectively. Docosanoic acid SLNs are thus considered as a promising carrier for intracellular bacterial treatment.

Composite Magnetite and Protein Containing CaCO3 Crystals. External Manipulation and Vaterite → Calcite Recrystallization-Mediated Release Performance.

PubMed

Sergeeva, Alena; Sergeev, Roman; Lengert, Ekaterina; Zakharevich, Andrey; Parakhonskiy, Bogdan; Gorin, Dmitry; Sergeev, Sergey; Volodkin, Dmitry

2015-09-30

Biocompatibility and high loading capacity of mesoporous CaCO3 vaterite crystals give an option to utilize the polycrystals for a wide range of (bio)applications. Formation and transformations of calcium carbonate polymorphs have been studied for decades, aimed at both basic and applied research interests. Here, composite multilayer-coated calcium carbonate polycrystals containing Fe3O4 magnetite nanoparticles and model protein lysozyme are fabricated. The structure of the composite polycrystals and vaterite → calcite recrystallization kinetics are studied. The recrystallization results in release of both loaded protein and Fe3O4 nanoparticles (magnetic manipulation is thus lost). Fe3O4 nanoparticles enhance the recrystallization that can be induced by reduction of the local pH with citric acid and reduction of the polycrystal crystallinity. Oppositely, the layer-by-layer assembled poly(allylamine hydrochloride)/poly(sodium styrenesulfonate) polyelectrolyte coating significantly inhibits the vaterite → calcite recrystallization (from hours to days) most likely due to suppression of the ion exchange giving an option to easily tune the release kinetics for a wide time scale, for example, for prolonged release. Moreover, the recrystallization of the coated crystals results in formulation of multilayer capsules keeping the feature of external manipulation. This study can help to design multifunctional microstructures with tailor-made characteristics for loading and controlled release as well as for external manipulation.

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.

PubMed

Kane, Lesley A; Yung, Christina K; Agnetti, Giulio; Neverova, Irina; Van Eyk, Jennifer E

2006-11-01

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.

Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes.

PubMed

Fong, Gloria; Backman, Ludvig J; Hart, David A; Danielson, Patrik; McCormack, Bob; Scott, Alex

2013-01-01

The loss of collagen organization is considered a hallmark histopathologic feature of tendinosis. At the cellular level, tenocytes have been shown to produce signal substances that were once thought to be restricted to neurons. One of the main neuropeptides implicated in tendinosis, substance P (SP), is known to influence collagen organization, particularly after injury. The aim of this study was to examine the influence of SP on collagen remodeling by primary human tendon cells cultured in vitro in three-dimensional collagen lattices. We found that SP stimulation led to an increased rate of collagen remodeling mediated via the neurokinin-1 receptor (NK-1 R), the preferred cell receptor for SP. Gene expression analysis showed that SP stimulation resulted in significant increases in MMP3, COL3A1 and ACTA2 mRNA levels in the collagen lattices. Furthermore, cyclic tensile loading of tendon cell cultures along with the administration of exogenous SP had an additive effect on MMP3 expression. Immunoblotting confirmed that SP increased MMP3 protein levels via the NK-1 R. This study indicates that SP, mediated via NK-1 R, increases collagen remodeling and leads to increased MMP3 mRNA and protein expression that is further enhanced by cyclic mechanical loading. Copyright © 2012 Orthopaedic Research Society.

Enhanced protective immune response to PCV2 subunit vaccine by co-administration of recombinant porcine IFN-γ in mice.

PubMed

Wang, Yi-Ping; Liu, Dan; Guo, Long-Jun; Tang, Qing-Hai; Wei, Yan-Wu; Wu, Hong-Li; Liu, Jian-Bo; Li, Sheng-Bin; Huang, Li-Ping; Liu, Chang-Ming

2013-01-21

The capsid (Cap) protein of PCV2 is the major immunogenic protein that is crucial to induce PCV2-specific neutralizing antibodies and protective immunity; thus, it is a suitable target antigen for the research and development of genetically engineered vaccines against PCV2 infection. IFN-γ has exhibited potential efficacy as an immune adjuvant that enhances the immunogenicity of certain vaccines in experimental animal models. In this study, three recombinant proteins: PCV2-Cap protein, porcine IFN-γ (PoIFN-γ), and the fusion protein (Cap-PoIFN-γ) of PCV2-Cap protein and PoIFN-γ were respectively expressed in the baculovirus system, and analyzed by Western blot and indirect ELISA. Additionally, we evaluated the enhancement of the protective immune response to the Cap protein-based PCV2 subunit vaccine elicited by co-administration of PoIFN-γ in mice. Vaccination of mice with the PCV2-Cap+PoIFN-γ vaccine elicited significantly higher levels of PCV2-specific IPMA antibodies, neutralizing antibodies, and lymphocyte proliferative responses compared to the Cap-PoIFN-γ vaccine, the PCV2-Cap vaccine, and LG-strain. Following virulent PCV2 challenge, no viraemia was detected in all immunized groups, and the viral loads in lungs of the PCV2-Cap+PoIFN-γ group were significantly lower compared to the Cap-PoIFN-γ group, the LG-strain group, and the mock group, but slightly lower compared to the PCV2-Cap group. These findings suggested that PoIFN-γ substantially enhanced the protective immune response to the Cap protein-based PCV2 subunit vaccine, and that the PCV2-Cap+PoIFN-γ subunit vaccine potentially serves as an attractive candidate vaccine for the prevention and control of PCV2-associated diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Physical and biological characterization of sericin-loaded copolymer liposomes stabilized by polyvinyl alcohol.

PubMed

Suktham, Kunat; Koobkokkruad, Thongchai; Saesoo, Somsak; Saengkrit, Nattika; Surassmo, Suvimol

2016-12-01

Sericin protein (SP) is widely used as a nutrient biomaterial for biomedical and cosmeceutical applications although it shows low stability to heat and light. To overcome these problems and add value to wastewater from the silk industry, sericin protein was recovered as sericin-loaded copolymer-liposomes (SP-PVA-LP), prepared through thin film hydration. The size and morphology of the liposomes were investigated using dynamic light scattering (DLS), and electron microscopy (SEM and TEM). The particle size, liposome surface morphology and encapsulation efficiency of SP were dependent on PVA concentration. The hydrodynamic size of the nanoparticles was between 200 and 400nm, with the degree of negative charge contingent on sericin loading. SEM and TEM images confirmed the mono-dispersity, and spherical nature of the particles, with FTIR measurements confirming the presence of surface bound PVA. Exposure of liposomes to 500ppm sericin highlighted a dependence of encapsulation efficiency on PVA content; 2% surface PVA proved the optimal level for sericin loading. Cytotoxicity and viability assays revealed that SP-loaded surface modified liposomes promote cellular attachment and proliferation of human skin fibroblasts without adverse toxic effects. Surface modified copolymer liposomes show high performance in maintaining structural stability, and promoting enhancements in the solubility and bio-viability of sericin. Taken together, these biocompatible constructs allow for effective controlled release, augmenting sericin activity and resulting in effective drug delivery systems. Copyright © 2016 Elsevier B.V. All rights reserved.

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

PubMed Central

Ray, Sujay; Bandaria, Jigar N.; Qureshi, Mohammad H.; Yildiz, Ahmet; Balci, Hamza

2014-01-01

Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K+, POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA’s access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na+, in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals. PMID:24516170

Preparation and characterization of PLGA-β-CD polymeric nanoparticles containing methotrexate and evaluation of their effects on T47D cell line.

PubMed

Gorjikhah, Fatemeh; Azizi Jalalian, Farid; Salehi, Roya; Panahi, Yunes; Hasanzadeh, Arash; Alizadeh, Effat; Akbarzadeh, Abolfazl; Davaran, Soodabeh

2017-05-01

Among all cancers that affect women, breast cancer has most mortality rate. It is essential to attain more safe and efficient anticancer drugs. Recent advances in medical nanotechnology and biotechnology have caused in novel improvements in breast and other cancer drug delivery. Methotrexate is an anticancer drug that prevents the dihydrofolate reductase enzyme, which inhibits in the formation of DNA, RNA and proteins which have poor water-solubility. For enhancing the solubility and stability of drugs in delivery systems, we used methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles. The PLGA- beta-cyclodextrin nanoparticles were synthesized by a double emulsion method and characterized with FT-IR and SEM. T47D breast cancer cell lines were treated with equal concentrations of methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles and free methotrexate. MTT assay confirmed that methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles enhanced cytotoxicity and drug delivery in T47D breast cancer cells. These results indicate that encapsulated drugs could be effective in controlled drug release for a sustained period would serve the purpose for long-term treatment of many diseases such as breast cancer.

Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover.

PubMed

Jin, Mingjie; Liu, Yanping; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh

2017-08-01

High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied rapid bioconversion with integrated recycle technology (RaBIT) and extractive ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h. Ethanol productivity was enhanced by two times and enzyme loading was reduced by 30%. Glucan and xylan conversions reached as high as 98% with an enzyme loading of as low as 8.4 mg protein per g glucan. The overall ethanol yield was 227 g ethanol/kg EA-CS (191 g ethanol/kg untreated CS). Biotechnol. Bioeng. 2017;114: 1713-1720. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Creation of energetic biothermite inks using ferritin liquid protein

NASA Astrophysics Data System (ADS)

Slocik, Joseph M.; McKenzie, Ruel; Dennis, Patrick B.; Naik, Rajesh R.

2017-04-01

Energetic liquids function mainly as fuels due to low energy densities and slow combustion kinetics. Consequently, these properties can be significantly increased through the addition of metal nanomaterials such as aluminium. Unfortunately, nanoparticle additives are restricted to low mass fractions in liquids because of increased viscosities and severe particle agglomeration. Nanoscale protein ionic liquids represent multifunctional solvent systems that are well suited to overcoming low mass fractions of nanoparticles, producing stable nanoparticle dispersions and simultaneously offering a source of oxidizing agents for combustion of reactive nanomaterials. Here, we use iron oxide-loaded ferritin proteins to create a stable and highly energetic liquid composed of aluminium nanoparticles and ferritin proteins for printing and forming 3D shapes and structures. In total, this bioenergetic liquid exhibits increased energy output and performance, enhanced dispersion and oxidation stability, lower activation temperatures, and greater processability and functionality.

ADAM10 Missense Mutations Potentiate β-Amyloid Accumulation by Impairing Prodomain Chaperone Function

PubMed Central

Suh, Jaehong; Choi, Se Hoon; Romano, Donna M.; Gannon, Moira A.; Lesinski, Andrea N.; Kim, Doo Yeon; Tanzi, Rudolph E.

2014-01-01

SUMMARY The generation of Aβ, the main component of senile plaques in Alzheimer’s disease (AD), is precluded by α-secretase cleavage within the Aβ domain of the amyloid precursor protein (APP). We identified two rare mutations (Q170H and R181G) in the prodomain of the metalloprotease, ADAM10, that co-segregate with late-onset AD (LOAD). Here, we addressed the pathogenicity of these mutations in transgenic mice expressing human ADAM10 in brain. In Tg2576 AD mice, both mutations attenuated α-secretase activity of ADAM10 and shifted APP processing toward β-secretase-mediated cleavage, while enhancing Aβ plaque load and reactive gliosis. We also demonstrated ADAM10 expression potentiates adult hippocampal neurogenesis, which is reduced by the LOAD mutations. Mechanistically, both LOAD mutations impaired the molecular chaperone activity of ADAM10 prodomain. Collectively, these findings suggest that diminished α-secretase activity, owing to LOAD ADAM10 prodomain mutations, leads to AD-related pathology, strongly supporting ADAM10 as a promising therapeutic target for this devastating disease. PMID:24055016

ADAM10 missense mutations potentiate β-amyloid accumulation by impairing prodomain chaperone function.

PubMed

Suh, Jaehong; Choi, Se Hoon; Romano, Donna M; Gannon, Moira A; Lesinski, Andrea N; Kim, Doo Yeon; Tanzi, Rudolph E

2013-10-16

The generation of Aβ, the main component of senile plaques in Alzheimer's disease (AD), is precluded by α-secretase cleavage within the Aβ domain of the amyloid precursor protein (APP). We identified two rare mutations (Q170H and R181G) in the prodomain of the metalloprotease, ADAM10, that cosegregate with late-onset AD (LOAD). Here, we addressed the pathogenicity of these mutations in transgenic mice expressing human ADAM10 in brain. In Tg2576 AD mice, both mutations attenuated α-secretase activity of ADAM10 and shifted APP processing toward β-secretase-mediated cleavage, while enhancing Aβ plaque load and reactive gliosis. We also demonstrated ADAM10 expression potentiates adult hippocampal neurogenesis, which is reduced by the LOAD mutations. Mechanistically, both LOAD mutations impaired the molecular chaperone activity of ADAM10 prodomain. Collectively, these findings suggest that diminished α-secretase activity, owing to LOAD ADAM10 prodomain mutations, leads to AD-related pathology, strongly supporting ADAM10 as a promising therapeutic target for this devastating disease. Copyright © 2013 Elsevier Inc. All rights reserved.

QUANTIFYING LOAD-INDUCED SOLUTE TRANSPORT AND SOLUTE-MATRIX INTERACTIONS WITHIN THE OSTEOCYTE LACUNAR-CANALICULAR SYSTEM

PubMed Central

Wang, Bin; Zhou, Xiaozhou; Price, Christopher; Li, Wen; Pan, Jun; Wang, Liyun

2012-01-01

Osteocytes, the most abundant cells in bone, are critical in maintaining tissue homeostasis and orchestrating bone’s mechanical adaptation. Osteocytes depend upon load-induced convection within the lacunar-canalicular system (LCS) to maintain viability and to sense their mechanical environment. Using the fluorescence recovery after photobleaching (FRAP) imaging approach, we previously quantified the convection of a small tracer (sodium fluorescein, 376Da) in the murine tibial LCS for an intermittent cyclic loading (Price et al., 2011. JBMR 26:277-85). In the present study we first expanded the investigation of solute transport using a larger tracer (parvalbumin, 12.3kDa), which is comparable in size to some signaling proteins secreted by osteocytes. Murine tibiae were subjected to sequential FRAP tests under rest-inserted cyclic loading while the loading magnitude (0, 2.8, or 4.8N) and frequency (0.5, 1, or 2 Hz) were varied. The characteristic transport rate k and the transport enhancement relative to diffusion (k/k0) were measured under each loading condition, from which the peak solute velocity in the LCS was derived using our LCS transport model. Both the transport enhancement and solute velocity increased with loading magnitude and decreased with loading frequency. Furthermore, the solute-matrix interactions, quantified in terms of the reflection coefficient through the osteocytic pericellular matrix (PCM), were measured and theoretically modeled. The reflection coefficient of parvalbumin (σ=0.084) was derived from the differential fluid and solute velocities within loaded bone. Using a newly developed PCM sieving model, the PCM’s fiber configurations accounting for the measured interactions were obtained for the first time. The present study provided not only new data on the micro-fluidic environment experienced by osteocytes in situ, but also a powerful quantitative tool for future study of the PCM, the critical interface that controls both outside-in and inside-out signaling in osteocytes during normal bone adaptation and in pathological conditions. PMID:23109140

Inclusion complex from cyclodextrin-grafted hyaluronic acid and pseudo protein as biodegradable nano-delivery vehicle for gambogic acid.

PubMed

Ji, Ying; Shan, Shuo; He, Mingyu; Chu, Chih-Chang

2017-10-15

β-Cyclodextrin can form inclusion complex with a series of guest molecules including phenyl moieties, and has gained considerable popularity in the study of supramolecular nanostructure. In this study, a biodegradable nanocomplex (HA(CD)-4Phe4 nanocomplex) was developed from β-cyclodextrin grafted hyaluronic acid (HA) and phenylalanine based poly(ester amide). The phenylalanine based poly(ester amide) is a biodegradable pseudo protein which provides the encapsulation capacity for gambogic acid (GA), a naturally-derived chemotherapeutic which has been effectively employed to treat multidrug resistant tumor. The therapeutic potency of free GA is limited due to its poor solubility in water and the lack of tumor-selective toxicity. The nanocomplex carrier enhanced the solubility and availability of GA in aqueous media, and the HA component enabled the targeted delivery to tumor cells with overexpression of CD44 receptors. In the presence of hyaluronidase, the release of GA from the nanocomplex was significantly accelerated, due to the enzymatic biodegradation of the carrier. Compared to free GA, GA-loaded nanocomplex exhibited improved cytotoxicity in MDA-MB-435/MDR multidrug resistant melanoma cells, and induced enhanced level of apoptosis and mitochondrial depolarization, at low concentration of GA (1-2µM). The nanocomplex enhanced the therapeutic potency of GA, especially when diluted in physiological environment. In addition, suppressed matrix metalloproteinase activity was also detected in MDA-MB-435/MDR cells treated by GA-loaded nanocomplex, which demonstrated its potency in the inhibition of tumor metastasis. The in vitro data suggested that HA(CD)-4Phe4 nanocomplex could provide a promising alternative in the treatment of multidrug resistant tumor cells. Gambogic acid (GA), naturally derived from genus Garcinia trees, exhibited significant cytotoxic activity against multiple types of tumors with resistance to traditional chemotherapeutics. Unfortunately, the poor solubility of GA in conventional pharmaceutical solvents and non-targeted distribution in normal tissues greatly limited its therapeutic potency. To overcome the challenges, we develop a nanoplatform from the supramolecular assembly of β-cyclodextrin grafted hyaluronic acid (HA) and phenylalanine based pseudo protein. The pseudo protein in the nanocomplex provided the hydrophobic interaction and loading capacity for GA, while the HA component targeted the overexpressed CD44 receptor and improved the selective endocytosis in multidrug resistant melanoma cells. The supramolecular nanocomplex provide a promising platform for the delivery of hydrophobic chemotherapeutics to improve the bioavailability and efficiency. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

PubMed

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

Evaluation of Doxorubicin-loaded 3-Helix Micelles as Nanocarriers

PubMed Central

Dube, Nikhil; Shu, Jessica Y.; Dong, He; Seo, Jai W.; Ingham, Elizabeth; Kheirolomoom, Azadeh; Chen, Pin-Yuan; Forsayeth, John; Bankiewicz, Krystof; Ferrara, Katherine W.; Xu, Ting

2013-01-01

Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt% doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics. PMID:24050265

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

PubMed Central

Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-01-01

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

PubMed

Juhász, Tamás; Szentléleky, Eszter; Somogyi, Csilla Szűcs; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-07-29

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

The Cu2+-nitrilotriacetic acid complex improves loading of α-helical double histidine site for precise distance measurements by pulsed ESR

NASA Astrophysics Data System (ADS)

Ghosh, Shreya; Lawless, Matthew J.; Rule, Gordon S.; Saxena, Sunil

2018-01-01

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand β-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu2+-NTA.

Targeted Doxorubicin-Loaded Bacterially Derived Nano-Cells for the Treatment of Neuroblastoma.

PubMed

Sagnella, Sharon M; Trieu, Jennifer; Brahmbhatt, Himanshu; MacDiarmid, Jennifer A; MacMillan, Alex; Whan, Renee M; Fife, Christopher M; McCarroll, Joshua A; Gifford, Andrew J; Ziegler, David S; Kavallaris, Maria

2018-05-01

Advanced stage neuroblastoma is an aggressive disease with limited treatment options for patients with drug-resistant tumors. Targeted delivery of chemotherapy for pediatric cancers offers promise to improve treatment efficacy and reduce toxicity associated with systemic chemotherapy. The EnGeneIC Dream Vector (EDV TM ) is a nanocell, which can package chemotherapeutic drugs and target tumors via attachment of bispecific proteins to the surface of the nanocell. Phase I trials in adults with refractory tumors have shown an acceptable safety profile. Herein we investigated the activity of EGFR-targeted and doxorubicin-loaded EDV TM ( EGFR EDV TM Dox ) for the treatment of neuroblastoma. Two independent neuroblastoma cell lines with variable expression of EGFR protein [SK-N-BE(2), high; SH-SY-5Y, low] were used. EGFR EDV TM Dox induced apoptosis in these cells compared to control, doxorubicin, or non-doxorubicin loaded EGFR EDV TM In three-dimensional tumor spheroids, imaging and fluorescence life-time microscopy revealed that EGFR EDV TM Dox had a marked enhancement of doxorubicin penetration compared to doxorubicin alone, and improved penetration compared to non-EGFR-targeted EDV TM Dox , with enhanced spheroid penetration leading to increased apoptosis. In two independent orthotopic human neuroblastoma xenograft models, short-term studies (28 days) of tumor-bearing mice led to a significant decrease in tumor size in EGFR EDV TM Dox -treated animals compared to control, doxorubicin, or non-EGFR EDV TM Dox There was increased TUNEL staining of tumors at day 28 compared to control, doxorubicin, or non-EGFR EDV TM Dox Moreover, overall survival was increased in neuroblastoma mice treated with EGFR EDV TM Dox ( P < 0007) compared to control. Drug-loaded bispecific-antibody targeted EDVs TM offer a highly promising approach for the treatment of aggressive pediatric malignancies such as neuroblastoma. Mol Cancer Ther; 17(5); 1012-23. ©2018 AACR . ©2018 American Association for Cancer Research.

Sucrose Transporter ZmSut1 Expression and Localization Uncover New Insights into Sucrose Phloem Loading1[OPEN

PubMed Central

Baker, R. Frank; Leach, Kristen A.; Boyer, Nathanial R.; Skopelitis, Tara; Jackson, David; Braun, David M.

2016-01-01

Sucrose transporters (SUTs) translocate sucrose (Suc) across cellular membranes, and in eudicots, multiple SUTs are known to function in Suc phloem loading in leaves. In maize (Zea mays), the Sucrose Transporter1 (ZmSut1) gene has been implicated in Suc phloem loading based upon RNA expression in leaves, electrophysiological experiments, and phenotypic analysis of zmsut1 mutant plants. However, no previous studies have examined the cellular expression of ZmSut1 RNA or the subcellular localization of the ZmSUT1 protein to assess the gene’s hypothesized function in Suc phloem loading or to evaluate its potential roles, such as phloem unloading, in nonphotosynthetic tissues. To this end, we performed RNA in situ hybridization experiments, promoter-reporter gene analyses, and ZmSUT1 localization studies to elucidate the cellular expression pattern of the ZmSut1 transcript and protein. These data showed that ZmSut1 was expressed in multiple cell types throughout the plant and indicated that it functions in phloem companion cells to load Suc and also in other cell types to retrieve Suc from the apoplasm to prevent its accumulation and loss to the transpiration stream. Additionally, by comparing a phloem-mobile tracer with ZmSut1 expression, we determined that developing maize leaves dynamically switch from symplasmic to apoplasmic phloem unloading, reconciling previously conflicting reports, and suggest that ZmSut1 does not have an apparent function in either unloading process. A model for the dual roles for ZmSut1 function (phloem loading and apoplasmic recycling), Sut1 evolution, and its possible use to enhance Suc export from leaves in engineering C3 grasses for C4 photosynthesis is discussed. PMID:27621426

Meal and beta-alanine coingestion enhances muscle carnosine loading.

PubMed

Stegen, Sanne; Blancquaert, Laura; Everaert, Inge; Bex, Tine; Taes, Youri; Calders, Patrick; Achten, Eric; Derave, Wim

2013-08-01

Beta-alanine (BA) is a popular ergogenic supplement because it can induce muscle carnosine loading. We hypothesize that, by analogy with creatine supplementation, 1) an inverse relationship between urinary excretion and muscle loading is present, and 2) the latter is stimulated by carbohydrate- and protein-induced insulin action. In study A, the effect of a 5-wk slow-release BA (SRBA) supplementation (4.8 g · d(-1)) on whole body BA retention was determined in seven men. We further determined whether the coingestion of carbohydrates and proteins with SRBA would improve retention. In study B (34 subjects), we explored the effect of meal timing on muscle carnosine loading (3.2 g · d(-1) during 6-7 wk). One group received pure BA (PBA) in between the meals; the other received PBA at the start of the meals, to explore the effect of meal-induced insulin release. Further, we compared with a third group receiving SRBA at the start of the meals. Orally ingested SRBA has a very high whole body retention (97%-98%) that is not declining throughout the 5-wk supplementation period, nor is it influenced by the coingestion of macronutrients. Thus, a very small portion (1%-2%) is lost through urinary excretion, and equally only a small portion is incorporated into muscle carnosine (≈ 3%), indicating that most ingested BA is metabolized (possibly through oxidation). Second, in soleus muscles, the efficiency of carnosine loading is significantly higher when PBA is coingested with a meal (+64%) compared with in between the meals (+41%), suggesting that insulin stimulates muscle carnosine loading. Finally, the chronic supplementation of SRBA versus PBA seems equally effective.

Preparation, characterization and immunological evaluation: canine parvovirus synthetic peptide loaded PLGA nanoparticles.

PubMed

Derman, Serap; Mustafaeva, Zeynep Akdeste; Abamor, Emrah Sefik; Bagirova, Melahat; Allahverdiyev, Adil

2015-10-20

Canine parvovirus 2 (CPV-2) remains a significant worldwide canine pathogen and the most common cause of viral enteritis in dogs. The 1 L15 and 7 L15 peptides overlap each other with QPDGGQPAV residues (7-15 of VP2 capsid protein of CPV) is shown to produce high immune response. PLGA nanoparticles were demonstrated to have special properties such as; controlled antigen release, protection from degradation, elimination of booster-dose and enhancing the cellular uptake by antigen presenting cells. Nevertheless, there is no study available in literature, about developing vaccine based on PLGA nanoparticles with adjuvant properties against CPV. Thus, the aim of the present study was to synthesize and characterize high immunogenic W-1 L19 peptide (from the VP2 capsid protein of CPV) loaded PLGA nanoparticle and to evaluate their in vitro immunogenic activity. PLGA nanoparticles were produced with 5.26 ± 0.05 % loading capacity and high encapsulation efficiency with 81.2 ± 3.1 %. Additionally, it was evaluated that free NPs and W-1 L19 peptide encapsulated PLGA nanoparticles have Z-ave of 183.9 ± 12.1 nm, 221.7 ± 15.8 nm and polydispersity index of 0.107 ± 0.08, 0.135 ± 0.12 respectively. It was determined that peptide loaded PLGA nanoparticles were successfully phagocytized by macrophage cells and increased NO production at 2-folds (*P < 0.05) in contrast to free peptide, and 3-folds (*P < 0.01) in contrast to control. In conclusion, for the first time, W-1 L19 peptide loaded PLGA nanoparticles were successfully synthesized and immunogenic properties evaluated. Obtained results showed that PLGA nanoparticles enhanced the capacity of W-1 L19 peptide to induce nitric oxide production in vitro due to its adjuvant properties. Depend on the obtained results, these nanoparticles can be accepted as potential vaccine candidate against Canine Parvovirus. Studies targeting PLGA nanoparticles based delivery system must be maintained in near future in order to develop new and more effective nano-vaccine formulations.

Self-Assembled Nanocarriers Based on Amphiphilic Natural Polymers for Anti- Cancer Drug Delivery Applications.

PubMed

Sabra, Sally; Abdelmoneem, Mona; Abdelwakil, Mahmoud; Mabrouk, Moustafa Taha; Anwar, Doaa; Mohamed, Rania; Khattab, Sherine; Bekhit, Adnan; Elkhodairy, Kadria; Freag, May; Elzoghby, Ahmed

2017-01-01

Micellization provides numerous merits for the delivery of water insoluble anti-cancer therapeutic agents including a nanosized 'core-shell' drug delivery system. Recently, hydrophobically-modified polysaccharides and proteins are attracting much attention as micelle forming polymers to entrap poorly soluble anti-cancer drugs. By virtue of their small size, the self-assembled micelles can passively target tumor tissues via enhanced permeation and retention effect (EPR). Moreover, the amphiphilic micelles can be exploited for active-targeted drug delivery by attaching specific targeting ligands to the outer micellar hydrophilic surface. Here, we review the conjugation techniques, drug loading methods, physicochemical characteristics of the most important amphiphilic polysaccharides and proteins used as anti-cancer drug delivery systems. Attention focuses on the mechanisms of tumor-targeting and enhanced anti-tumor efficacy of the encapsulated drugs. This review will highlight the remarkable advances of hydrophobized polysaccharide and protein micelles and their potential applications as anti-cancer drug delivery nanosystems. Micellar nanocarriers fabricated from amphiphilic natural polymers hold great promise as vehicles for anti-cancer drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Sensitivity of the appetite control system in obese subjects to nutritional and serotoninergic challenges.

PubMed

Hill, A J; Blundell, J E

1990-03-01

The sensitivity of the appetite system of a group of obese individuals was assessed in response to two challenges known to reduce hunger and enhance satiety in lean people. The challenges were the presentation of a caloric (high protein) load and the activation of serotonin systems. Eight obese female adults (BMI = 38) received 2 X 15 mg d-fenfluramine or placebo daily for 3 days, the study conforming to a 2 X 2 factor (drug X lunch type), double blind, repeated measures design. Three hours after dosing on day 3 they ate either a high carbohydrate (63 percent of total energy) or high protein (54 percent) lunchtime meal (the caloric load). These fixed meal challenges were equal in energy (475 kcal), weight and fat content. Ratings of hunger motivation and food preferences were tracked over the course of lunch and for a further 3 hours, at which point subjects returned for a self-selection test meal. Intakes from this second open meal revealed significant main effects of both caloric load and drug on energy intake, with the high protein d-fenfluramine combination being the most potent anorectic pairing. These findings were supported by the profiles of hunger motivation. This study has confirmed that the appetite system of these subjects was responsive to these biologically relevant challenges. The results suggest that the combination of an appetite modulating drug with specific dietary intervention may represent an effective strategy for the management of hunger arising from caloric restriction.

Creation of energetic biothermite inks using ferritin liquid protein

PubMed Central

Slocik, Joseph M.; McKenzie, Ruel; Dennis, Patrick B.; Naik, Rajesh R.

2017-01-01

Energetic liquids function mainly as fuels due to low energy densities and slow combustion kinetics. Consequently, these properties can be significantly increased through the addition of metal nanomaterials such as aluminium. Unfortunately, nanoparticle additives are restricted to low mass fractions in liquids because of increased viscosities and severe particle agglomeration. Nanoscale protein ionic liquids represent multifunctional solvent systems that are well suited to overcoming low mass fractions of nanoparticles, producing stable nanoparticle dispersions and simultaneously offering a source of oxidizing agents for combustion of reactive nanomaterials. Here, we use iron oxide-loaded ferritin proteins to create a stable and highly energetic liquid composed of aluminium nanoparticles and ferritin proteins for printing and forming 3D shapes and structures. In total, this bioenergetic liquid exhibits increased energy output and performance, enhanced dispersion and oxidation stability, lower activation temperatures, and greater processability and functionality. PMID:28447665

Antiglioma activity of curcumin-loaded lipid nanoparticles and its enhanced bioavailability in brain tissue for effective glioblastoma therapy.

PubMed

Kundu, Paromita; Mohanty, Chandana; Sahoo, Sanjeeb K

2012-07-01

Glioblastoma, the most aggressive form of brain and central nervous system tumours, is characterized by high rates proliferation, migration and invasion. The major road block in the delivery of drugs to the brain is the blood-brain barrier, along with the expression of various multi-drug resistance (MDR) proteins that cause the efflux of a wide range of chemotherapeutic drugs. Curcumin, a herbal drug, is known to inhibit cellular proliferation, migration and invasion and induce apoptosis of glioma cells. It also has the potential to modulate MDR in glioma cells. However, the greatest challenge in the administration of curcumin stems from its low bioavailability and high rate of metabolism. To circumvent the above pitfalls of curcumin we have developed curcumin-loaded glyceryl monooleate (GMO) nanoparticles (NP) coated with the surfactant Pluronic F-68 and vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for brain delivery. We demonstrated that our curcumin-loaded NPs inhibit cellular proliferation, migration and invasion along with a higher percentage of cell cycle arrest and telomerase inhibition, thus leading to a greater percentage apoptotic cell death in glioma cells compared with native curcumin. An in vivo study demonstrated enhanced bioavailability of curcumin in blood serum and brain tissue when delivered by curcumin-loaded GMO NPs compared with native curcumin in a rat model. Thus, curcumin-loaded GMO NPs can be used as an effective delivery system to overcome the challenges of drug delivery to the brain, providing a new approach to glioblastoma therapy. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ingestion of soy-whey blended protein augments sports performance and ameliorates exercise-induced fatigue in a rat exercise model.

PubMed

Ren, Guangxu; Yi, Suqing; Zhang, Hongru; Wang, Jing

2017-02-22

This study sought to determine the effects of soy-whey blended protein supplementation on sports performance and related biochemical parameters after long-term training. After a week of adaptation, eighteen 6-week-old male Wistar rats were randomly assigned to 3 groups: the standard chow diet plus whey protein (Whey) group, the standard chow diet plus soy-whey blended protein (BP) group and the standard chow diet only (control) group. Each group included 6 rats for the seven-week experiment. Before the experiment, the baseline values of body weight, grasping force and time to exhaustion due to the loaded-swimming test were recorded for each group. During the experimental period, all rats performed the loaded-swimming test until exhaustion five days each week. The results showed that the mean maximum grasping force of the BP group significantly increased between the 5 th and the 7 th week (p < 0.05) compared with the other groups. The ingestion of blended protein for 7 weeks significantly increased the mean time to exhaustion due to swimming by 1.5-fold and 1.2-fold compared with the control and Whey groups, respectively. The plasma levels of leucine, isoleucine and valine were significantly higher at 60 min after the blended protein intervention compared with the Whey and control interventions (p < 0.05). Furthermore, the ingestion of soy-whey blended protein enhanced the activities of lactate dehydrogenase and superoxide dismutase and decreased the levels of malondialdehyde in serum. These results collectively suggest that soy-whey blended protein ingestion with resistance exercise can improve sports performance and ameliorate exercise-induced fatigue in rats.

Thin-walled SnO2 nanotubes functionalized with Pt and Au catalysts via the protein templating route and their selective detection of acetone and hydrogen sulfide molecules

NASA Astrophysics Data System (ADS)

Jang, Ji-Soo; Kim, Sang-Joon; Choi, Seon-Jin; Kim, Nam-Hoon; Hakim, Meggie; Rothschild, Avner; Kim, Il-Doo

2015-10-01

Bio-inspired Pt (~2 nm) and Au (~2.7 nm) catalysts encapsulated by a protein shell, i.e., Pt-apoferritin (Pt@AF) and Au-apoferriten (Au@AF), were synthesized via the hollow protein nanocage (apoferritin) templating route and directly functionalized on the interior and exterior walls of electrospun SnO2 nanotubes (NTs) during controlled single-nozzle electrospinning followed by high temperature calcination with heating rate control. Fast crystallization of the exterior shell and outward diffusion of the interior Sn precursors and crystallites result in the continued growth of a tubular wall, which is related to rapid heating driven Ostwald-ripening behavior. Very importantly, the Pt and Au nanoparticles (NPs) were immobilized onto thin-walled SnO2 NTs with a diameter of ~350 nm and a shell thickness of ~40 nm without any aggregation of catalysts due to high dispersibility, which originated from repulsive electrostatic (Coulombic) forces acting on the surface charged protein shells, leading to an enhanced catalytic effect and outstanding gas sensing properties. Pt-loaded SnO2 NTs exhibited superior acetone response (Rair/Rgas = 92 at 5 ppm) compared to pure SnO2 NFs (Rair/Rgas = 4.8 at 5 ppm) and SnO2 NTs (Rair/Rgas = 11 at 5 ppm) while Au-loaded SnO2 NTs showed a high response when exposed to hydrogen sulfide (Rair/Rgas = 34 at 5 ppm), offering selective gas detection with minimal cross-sensitivity against other interfering gases such as NH3, CO, NO, C6H5CH3, and C5H12. Our results provide a new insight into facile, cost-effective, and highly dispersible catalyst loading on the interior and exterior walls of hollow metal oxide NTs via simple electrospinning as a potential breath analyzer.Bio-inspired Pt (~2 nm) and Au (~2.7 nm) catalysts encapsulated by a protein shell, i.e., Pt-apoferritin (Pt@AF) and Au-apoferriten (Au@AF), were synthesized via the hollow protein nanocage (apoferritin) templating route and directly functionalized on the interior and exterior walls of electrospun SnO2 nanotubes (NTs) during controlled single-nozzle electrospinning followed by high temperature calcination with heating rate control. Fast crystallization of the exterior shell and outward diffusion of the interior Sn precursors and crystallites result in the continued growth of a tubular wall, which is related to rapid heating driven Ostwald-ripening behavior. Very importantly, the Pt and Au nanoparticles (NPs) were immobilized onto thin-walled SnO2 NTs with a diameter of ~350 nm and a shell thickness of ~40 nm without any aggregation of catalysts due to high dispersibility, which originated from repulsive electrostatic (Coulombic) forces acting on the surface charged protein shells, leading to an enhanced catalytic effect and outstanding gas sensing properties. Pt-loaded SnO2 NTs exhibited superior acetone response (Rair/Rgas = 92 at 5 ppm) compared to pure SnO2 NFs (Rair/Rgas = 4.8 at 5 ppm) and SnO2 NTs (Rair/Rgas = 11 at 5 ppm) while Au-loaded SnO2 NTs showed a high response when exposed to hydrogen sulfide (Rair/Rgas = 34 at 5 ppm), offering selective gas detection with minimal cross-sensitivity against other interfering gases such as NH3, CO, NO, C6H5CH3, and C5H12. Our results provide a new insight into facile, cost-effective, and highly dispersible catalyst loading on the interior and exterior walls of hollow metal oxide NTs via simple electrospinning as a potential breath analyzer. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04487a

Induction of Fos expression in the rat forebrain after intragastric administration of monosodium L-glutamate, glucose and NaCl.

PubMed

Otsubo, H; Kondoh, T; Shibata, M; Torii, K; Ueta, Y

2011-11-24

l-glutamate, an umami taste substance, is a key molecule coupled to a food intake signaling pathway. Furthermore, recent studies have unveiled new roles for dietary glutamate on gut-brain axis communication via activation of gut glutamate receptors and subsequent vagus nerve. In the present study, we mapped activation sites of the rat forebrain after intragastric load of 60 mM monosodium l-glutamate (MSG) by measurement of Fos protein, a functional marker of neuronal activation. The same concentration of d-glucose (sweet) and NaCl (salty) was used as controls. MSG administration exclusively produced enhanced Fos expression in four hypothalamic regions (the medial preoptic area, lateral hypothalamic area, dorsomedial nucleus, and arcuate nucleus). On the other hand, glucose administration exclusively enhanced Fos induction in the nucleus accumbens. Both MSG and glucose enhanced Fos induction in three brain regions (the habenular nucleus, paraventricular nucleus, and central nucleus of the amygdala). However, MSG induced Fos inductions were more potent than those of glucose in the habenular nucleus and paraventricular nucleus. Importantly, the present study identified for the first time two brain areas (the paraventricular and arcuate hypothalamic nuclei) that are more potently activated by intragastric MSG loads compared with glucose and NaCl. Overall, our results suggest significant activation of a neural network comprising the habenular nucleus, amygdala, and the hypothalamic subnuclei following intragastric load with glutamate. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Exosomes Secreted under Hypoxia Enhance Invasiveness and Stemness of Prostate Cancer Cells by Targeting Adherens Junction Molecules

PubMed Central

Ramteke, Anand; Ting, Harold; Agarwal, Chapla; Mateen, Samiha; Somasagara, Ranganathan; Hussain, Anowar; Graner, Michael; Frederick, Barbara; Agarwal, Rajesh; Deep, Gagan

2015-01-01

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2) or normoxic (21% O2) conditions, and exosomes secreted under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that ExoHypoxic have smaller average size as compared to ExoNormoxic. Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70) and Annexin II in ExoHypoxic compared to ExoNormoxic. Co-culturing with ExoHypoxic increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. ExoHypoxic also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to ExoNormoxic, ExoHypoxic showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in ExoHypoxic (160 proteins) compared to ExoNormoxic (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, ExoHypoxic targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that ExoHypoxic are loaded with unique proteins that could enhance invasiveness, stemness and induce microenvironment changes; thereby, promoting PCA aggressiveness. PMID:24347249

Functional reconstruction of critical-sized load-bearing bone defects using a Sclerostin-targeting miR-210-3p-based construct to enhance osteogenic activity.

PubMed

Hu, Bin; Li, Yan; Wang, Mohan; Zhu, Youming; Zhou, Yong; Sui, Baiyan; Tan, Yu; Ning, Yujie; Wang, Jie; He, Jiacai; Yang, Chi; Zou, Duohong

2018-06-10

A considerable amount of research has focused on improving regenerative therapy strategies for repairing defects in load-bearing bones. The enhancement of tissue regeneration with microRNAs (miRNAs) is being developed because miRNAs can simultaneously regulate multiple signaling pathways in an endogenous manner. In this study, we developed a miR-210-based bone repair strategy. We identified a miRNA (miR-210-3p) that can simultaneously up-regulate the expression of multiple key osteogenic genes in vitro. This process resulted in enhanced bone formation in a subcutaneous mouse model with a miR-210-3p/poly-L-lactic acid (PLLA)/bone marrow-derived stem cell (BMSC) construct. Furthermore, we constructed a model of critical-sized load-bearing bone defects and implanted a miR-210-3p/β-tricalcium phosphate (β-TCP)/bone mesenchymal stem cell (BMSC) construct into the defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. We also identified a new mechanism by which miR-210-3p regulates Sclerostin protein levels. This miRNA-based strategy may yield novel therapeutic methods for the treatment of regenerative defects in vital load-bearing bones by utilizing miRNA therapy for tissue engineering. The destroyed maxillofacial bone reconstruction is still a real challenge for maxillofacial surgeon, due to that functional bone reconstruction involved load-bearing. Base on the above problem, this paper developed a novel miR-210-3p/β-tricalcium phosphate (TCP)/bone marrow-derived stem cell (BMSC) construct (miR-210-3p/β-TCP/BMSCs), which lead to functional reconstruction of critical-size mandible bone defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. In addition, we also found the mechanism of how the delivered microRNA activated the signaling pathways of endogenous stem cells, leading to the defect regeneration. This miRNA-based strategy can be used to regenerate defects in vital load-bearing bones, thus addressing a critical challenge in regenerative medicine by utilizing miRNA therapy for tissue engineering. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Loading of halloysite nanotubes with BSA, α-Lac and β-Lg: a Fourier transform infrared spectroscopic and thermogravimetric study

NASA Astrophysics Data System (ADS)

Duce, Celia; Della Porta, Valentina; Bramanti, Emilia; Campanella, Beatrice; Spepi, Alessio; Tiné, Maria Rosaria

2017-02-01

Halloysite nanotubes (HNTs) are considered as ideal materials for biotechnological and medical applications. An important feature of halloysite is that it has a different surface chemistry on the inner and outer sides of the tubes. This property means that negatively-charged molecules can be selectively loaded inside the halloysite nanoscale its lumen. Loaded HNTs can be used for the controlled or sustained release of proteins, drugs, bioactive molecules and other agents. We studied the interaction between HNTs and bovine serum albumin, α lactalbumin and β -lactoglobulin loaded into HTNs using Fourier transform infrared spectroscopy and thermogravimetry. These techniques enabled us to study the protein conformation and thermal stability, respectively, and to estimate the amount of protein loaded into the HNTs. TEM images confirmed the loading of proteins into HTNs.

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

PubMed Central

Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

2014-01-01

Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. PMID:25364253

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles.

PubMed

Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

2014-01-01

Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects

PubMed Central

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m2/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration. PMID:25609957

THE ROLE OF MECHANOBIOLOGY IN TENDON HEALING

PubMed Central

Killian, Megan L.; Cavinatto, Leonardo; Galatz, Leesa M.; Thomopoulos, Stavros

2011-01-01

Mechanical cues affect tendon healing, homeostasis, and development in a variety of settings. Alterations in the mechanical environment are known to result in changes in the expression of extracellular matrix proteins, growth factors, transcription factors, and cytokines that can alter tendon structure and cell viability. Loss of muscle force in utero or in the immediate postnatal period delays tendon and enthesis development. The response of healing tendons to mechanical load varies depending on anatomic location. Flexor tendons require motion to prevent adhesion formation, yet excessive force results in gap formation and subsequent weakening of the repair. Excessive motion in the setting of anterior cruciate ligament reconstruction causes accumulation of macrophages, which are detrimental to tendon graft healing. Complete removal of load is detrimental to rotator cuff healing, yet large forces are also harmful. Controlled loading can enhance healing in most settings; however, a fine balance must be reached between loads that are too low (leading to a catabolic state) and too high (leading to micro-damage). This review will summarize existing knowledge of the mechanobiology of tendon development, homeostasis, and healing. PMID:22244066

Gelatin modified lipid nanoparticles for anti- viral drug delivery.

PubMed

K S, Joshy; S, Snigdha; Kalarikkal, Nandakumar; Pothen, Laly A; Thomas, Sabu

2017-10-01

The major challenges to clinical application of zidovudine are its moderate aqueous solubility and relative short half-life and serious side effects due to frequent administrations. We investigated the preparation of zidovudine-loaded nanoparticles based on lipids which were further modified with the polymer gelatin. Formulation and stability of the modified nanoparticles were analysed from the physico-chemical characterizations. The interactions of nanoparticles with blood components were tested by haemolysis and aggregation studies. The drug content and entrapment efficiencies were assessed by UV analysis. The effect of nanoparticles on protein adsorption was assessed by native polyacrylamide gel electrophoresis (PAGE). In vitro release studies showed a sustained release profile of zidovudine. In vitro cytotoxicity and cellular uptake of the zidovudine-loaded nanoparticles were performed in MCF-7 and neuro 2a brain cells. The enhanced cellular internalization of drug loaded modified nanoparticles in both the cell lines were revealed by fluorescence microscopy. Hence the present study focuses on the feasibility of zidovudine-loaded polymer modified lipid nanoparticles as carriers for safe and efficient HIV/AIDS therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphate binding reduces aortic angiotensin-converting enzyme and enhances nitric oxide bioactivity in experimental renal insufficiency.

PubMed

Eräranta, Arttu; Törmänen, Suvi; Kööbi, Peeter; Vehmas, Tuija I; Lakkisto, Päivi; Tikkanen, Ilkka; Moilanen, Eeva; Niemelä, Onni; Mustonen, Jukka; Pörsti, Ilkka

2014-01-01

Disturbed calcium-phosphorus metabolism is associated with increased kidney angiotensin-converting enzyme (ACE) in experimental chronic renal insufficiency (CRI). However, information about the effects of phosphate binding and loading on vascular ACE is lacking. Fifteen weeks after 5/6 nephrectomy (NX), rats were placed on a phosphate-binding (NX+Ca, 3.0% Ca), phosphate-loading (NX+Pi, 1.5% Pi), or control diet for 12 weeks (NX and sham). Aortic ACE, blood pressure, plasma phosphate, and parathyroid hormone were increased in the NX and NX+Pi groups, but were reduced with phosphate binding. Endothelium-mediated relaxations of isolated mesenteric conduit artery rings to acetylcholine were impaired in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. Experiments with nitric oxide (NO) synthase inhibition in vitro suggested that the NO-mediated component of acetylcholine response was lower in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. In all NX groups, aortic endothelial NO synthase (eNOS) was reduced, while plasma and urine concentrations of NO metabolites were increased. Aortic nitrated proteins and calcification were increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups. Hypertension in the NX model of CRI was associated with reduced vasorelaxation, decreased eNOS, and increased ACE and nitrated proteins in the aorta. Phosphate binding with calcium carbonate enhanced vasorelaxation via endogenous NO and suppressed elevation of ACE and nitrated proteins, suggesting reduced vascular oxidative stress. Our findings support the view that correction of the calcium-phosphorus balance prevents CRI-induced vascular pathophysiology.

Enhancement of bioavailability by formulating rhEPO ionic complex with lysine into PEG-PLA micelle

NASA Astrophysics Data System (ADS)

Shi, Yanan; Sun, Fengying; Wang, Dan; Zhang, Renyu; Dou, Changlin; Liu, Wanhui; Sun, Kaoxiang; Li, Youxin

2013-10-01

A composite micelle of ionic complex encapsulated into poly(ethylene glycol)-poly( d, l-lactide) (PEG-PLA) di-block copolymeric micelles was used for protein drug delivery to improve its pharmacokinetic performance. In this study, recombinant human erythropoietin (rhEPO, as a model protein) was formulated with lysine into composite micelles at a diameter of 71.5 nm with narrow polydispersity indices (PDIs < 0.3). Only a trace amount of protein was in aggregate form. The zeta potential of the spherical micelles was ranging from -0.54 to 1.39 mv, and encapsulation efficiency is high (80 %). The stability of rhEPO was improved significantly in composite micelles in vitro. Pharmacokinetic studies in rats showed significant, enhanced plasma retention of the composite micelles in comparison with native rhEPO. Areas under curve (AUCs) of the rhEPO released from the composite micelles were 4.5- and 2.3-folds higher than those of the native rhEPO and rhEPO-loaded PEG-PLA micelle, respectively. In addition, the composite micelles exhibited good biocompatibility using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay with human embryonic kidney (HEK293T) cells. All these features are preferable for utilizing the composite micelles as a novel protein delivery system.

Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica/chitosan hybrid for enhanced bone regeneration.

PubMed

Jung, Hyun-Do; Yook, Se-Won; Han, Cheol-Min; Jang, Tae-Sik; Kim, Hyoun-Ee; Koh, Young-Hag; Estrin, Yuri

2014-07-01

Porous Ti has been widely investigated for orthopedic and dental applications on account of their ability to promote implant fixation via bone ingrowth into pores. In this study, highly aligned porous Ti scaffolds coated with a bone morphogenetic protein (BMP)-loaded silica/chitosan hybrid were produced, and their bone regeneration ability was evaluated by in vivo animal experiments. Reverse freeze casting allowed for the creation of highly aligned pores, resulting in a high compressive strength of 254 ± 21 MPa of the scaffolds at a porosity level of ∼51 vol %. In addition, a BMP-loaded silica/chitosan hybrid coating layer with a thickness of ∼1 μm was uniformly deposited on the porous Ti scaffold, which enabled the sustained release of the BMP over a prolonged period of time up to 26 days. The cumulative amount of the BMP released was ∼4 μg, which was much higher than that released from the specimen without a hybrid coating layer. In addition, the bone regeneration ability of the porous Ti scaffold with a BMP-loaded silica/chitosan coating layer was examined by in vivo animal testing using a rabbit calvarial defect model and compared with those of the as-produced porous Ti scaffold and porous Ti scaffold with a silica/chitosan coating layer. After 4 weeks of healing, the specimen coated with a BMP-loaded silica/chitosan hybrid showed a much higher bone regeneration volume (∼36%) than the as-produced specimen (∼15%) (p < 0.005) and even the specimen coated with a silica/chitosan hybrid (∼25%) (p < 0.05). © 2013 Wiley Periodicals, Inc.

Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2

PubMed Central

Kang, Yunqing; Kim, Sungwoo; Khademhosseini, Ali; Yang, Yunzhi

2011-01-01

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading. PMID:21632105

Biomimetic hydrogel loaded with silk and l-proline for tissue engineering and wound healing applications.

PubMed

Thangavel, Ponrasu; Ramachandran, Balaji; Kannan, Ramya; Muthuvijayan, Vignesh

2017-08-01

The aim of this article was to develop silk protein (SF) and l-proline (LP) loaded chitosan-(CS) based hydrogels via physical cross linking for tissue engineering and wound healing applications. Silk fibroin, a biodegradable and biocompatible protein, and l-proline, an important imino acid that is required for collagen synthesis, were added to chitosan to improve the wound healing properties of the hydrogel. Characterization of these hydrogels revealed that CS/SF/LP hydrogels were blended properly and LP incorporated hydrogels showed excellent thermal stability and good surface morphology. Swelling study showed the water holding efficiency of the hydrogels to provide enough moisture at the wound surface. In vitro biodegradation results demonstrated that the hydrogels had good degradation rate in PBS with lysozyme. LP loaded hydrogels showed approximately a twofold increase in antioxidant activity. In vitro cytocompatibility studies using NIH 3T3 L1 cells showed increased cell viability (p < 0.01), migration, proliferation and wound healing activity (p < 0.001) in LP loaded hydrogels compared to CS and CS/SF hydrogels. Cell adhesion on SF and LP hydrogels were observed using SEM and compared to CS hydrogel. LP incorporation showed 74-78% of wound closure compared to 35% for CS/SF and 3% for CS hydrogels at 48 h. These results suggest that incorporation of LP can significantly accelerate wound healing process compared to pure CS and SF-loaded CS hydrogels. Hence, CS/LP hydrogels could be a potential wound dressing material for the enhanced wound tissue regeneration and repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1401-1408, 2017. © 2016 Wiley Periodicals, Inc.

Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells.

PubMed

Liang, Gaofeng; Kan, Shu; Zhu, Yanliang; Feng, Shuying; Feng, Wenpo; Gao, Shegan

2018-01-01

Exosomes are closed-membrane nanovesicles that are secreted by a variety of cells and exist in most body fluids. Recent studies have demonstrated the potential of exosomes as natural vehicles that target delivery of functional small RNA and chemotherapeutics to diseased cells. In this study, we introduce a new approach for the targeted delivery of exosomes loaded with functional miR-26a to scavenger receptor class B type 1-expressing liver cancer cells. The tumor cell-targeting function of these engineered exosomes was introduced by expressing in 293T cell hosts, the gene fusion between the transmembrane protein of CD63 and a sequence from Apo-A1. The exosomes harvested from these 293T cells were loaded with miR-26a via electroporation. The engineered exosomes were shown to bind selectively to HepG2 cells via the scavenger receptor class B type 1-Apo-A1 complex and then internalized by receptor-mediated endocytosis. The release of miR-26a in exosome-treated HepG2 cells upregulated miR-26a expression and decreased the rates of cell migration and proliferation. We also presented evidence that suggest cell growth was inhibited by miR-26a-mediated decreases in the amounts of key proteins that regulate the cell cycle. Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of miRNA-loaded exosomes that recognize specific biomarkers on the tumor cell.

Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hong; Yang, Yanling; Li, Yuxin

2015-02-06

Development of high resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse phase column (100 μm x 150 cm) coupled with Q Exactive MS. The column was capable of achieving a peak capacity of approximately 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was about 6 micrograms of peptides, although the column allowed loading as many as 20 micrograms. Gas phasemore » fractionation of peptide ions further increased the number of peptide identification by ~10%. Moreover, the combination of basic pH LC pre-fractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a postmortem brain sample of Alzheimer’s disease. As deep RNA sequencing of the same specimen suggested that ~16,000 genes were expressed, current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.« less

Subcritical Water Technology for Enhanced Extraction of Biochemical Compounds from Chlorella vulgaris

PubMed Central

Awaluddin, S. A.; Thiruvenkadam, Selvakumar; Izhar, Shamsul; Hiroyuki, Yoshida; Danquah, Michael K.; Harun, Razif

2016-01-01

Subcritical water extraction (SWE) technology has been used for the extraction of active compounds from different biomass materials with low process cost, mild operating conditions, short process times, and environmental sustainability. With the limited application of the technology to microalgal biomass, this work investigates parametrically the potential of subcritical water for high-yield extraction of biochemicals such as carbohydrates and proteins from microalgal biomass. The SWE process was optimized using central composite design (CCD) under varying process conditions of temperature (180–374°C), extraction time (1–20 min), biomass particulate size (38–250 μm), and microalgal biomass loading (5–40 wt.%). Chlorella vulgaris used in this study shows high volatile matter (83.5 wt.%) and carbon content (47.11 wt.%), giving advantage as a feedstock for biofuel production. The results showed maximum total carbohydrate content and protein yields of 14.2 g/100 g and 31.2 g/100 g, respectively, achieved under the process conditions of 277°C, 5% of microalgal biomass loading, and 5 min extraction time. Statistical analysis revealed that, of all the parameters investigated, temperature is the most critical during SWE of microalgal biomass for protein and carbohydrate production. PMID:27366748

Enhancement of fermentable sugar yield by competitive adsorption of non-enzymatic substances from yeast and cellulase on lignin.

PubMed

Tang, Yong; Lei, Fuhou; Cristhian, Carrasco; Liu, Zuguang; Yu, Hailong; Jiang, Jianxin

2014-03-20

Enhancement of enzymatic digestibility by some supplementations could reduce enzyme loading and cost, which is still too high to realize economical production of lignocellulosic biofuels. A recent study indicates that yeast hydrolysates (YH) have improved the efficiency of cellulases on digestibility of furfural residues (FR). In the current work, the components of YH were separated by centrifugation and size exclusion chromatography and finally characterized in order to better understand this positive effect. A 60.8% of nitrogen of yeast cells was remained in the slurry (YHS) after hydrothermal treatment. In the supernatant of YH (YHL), substances of high molecular weight were identified as proteins and other UV-absorbing compounds, which showed close molecular weight to components of cellulases. Those substances attributed to a synergetic positive effect on enzymatic hydrolysis of FR. The fraction of YHL ranged from 1.19 to 2.19 mL (elution volume) contained over 50% of proteins in YHL and had the best performance in stimulating the release of glucose. Experiment results proved the adsorption of proteins in YHL on lignin. Supplementation of cellulases with YH enhances enzymatic digestibility of FR mainly by a competitive adsorption of non-enzymatic substances on lignin. The molecular weight of these substances has a significant impact on their performance. Different strategies can be used for a good utilization of yeast cells in terms of biorefinery concept.

Formulation and in vitro characterization of protein-loaded liposomes

NASA Astrophysics Data System (ADS)

Kuzimski, Lauren

Background/Objective: Protein-based drugs are increasingly used to treat a variety of conditions including cancer and cardio-vascular disease. Due to the immune system's innate ability to degrade the foreign particles quickly, protein-based treatments are generally short-lived. To address this limitation, the objective of the study was to: 1) develop protein-loaded liposomes; 2) characterize size, stability, encapsulation efficiency and rate of protein release; and 3) determine intracellular uptake and distribution; and 4) protein structural changes. Method: Liposomes were loaded with a fluorescent-albumin using freeze-thaw (F/T) methodology. Albumin encapsulation and release were quantified by fluorescence spectroscopic techniques. Flow cytometry was used to determine liposome uptake by macrophages. Epifluorescence microscopy was used to determine cellular distribution of liposomes. Stability was determined using dynamic light scattering by measuring liposome size over one month period. Protein structure was determined using circular dichroism (CD). Result: Encapsulation of albumin in liposome was ˜90% and was dependent on F/T rates, with fifteen cycles yielding the highest encapsulation efficacy (p < 0.05). Albumin-loaded liposomes demonstrated consistent size (<300nm). Release of encapsulated albumin in physiological buffer at 25°C was ˜60% in 72 h. Fluorescence imaging suggested an endosomal route of cellular entry for the FITC-albumin liposome with maximum uptake rates in immune cells (30% at 2hour incubation). CD suggested protein structure is minimally impacted by freeze-thaw methodology. Conclusion: Using F/T as a loading method, we were able to successfully achieve a protein-loaded liposome that was under 300nm, had encapsulation of ˜90%. Synthesized liposomes demonstrated a burst release of encapsulate protein (60%) at 72 hours. Cellular trafficking confirmed endosomal uptake, and minimal protein damage was noticed in CD.

Viral load and clinical disease enhancement associated with a lentivirus cytotoxic T lymphocyte vaccine regimen

PubMed Central

Mealey, Robert H.; Leib, Steven R.; Littke, Matt H.; Wagner, Bettina; Horohov, David W.; McGuire, Travis C.

2009-01-01

Effective DNA-based vaccines against lentiviruses will likely induce CTL against conserved viral proteins. Equine infectious anemia virus (EIAV) infects horses worldwide, and serves as a useful model for lentiviral immune control. Although attenuated live EIAV vaccines have induced protective immune responses, DNA-based vaccines have not. In particular, DNA-based vaccines have had limited success in inducing CTL responses against intracellular pathogens in the horse. We hypothesized that priming with a codon-optimized plasmid encoding EIAV Gag p15/p26 with co-administration of a plasmid encoding an equine IL-2/IgG fusion protein as a molecular adjuvant, followed by boosting with a vaccinia vector expressing Gag p15/p26, would induce protective Gag-specific CTL responses. Although the regimen induced Gag-specific CTL in four of seven vaccinated horses, CTL were not detected until after the vaccinia boost, and protective effects were not observed in EIAV challenged vaccinates. Unexpectedly, vaccinates had significantly higher viral loads and more severe clinical disease, associated with the presence of vaccine-induced CTL. It was concluded that 1.) further optimization of the timing and route of DNA immunization was needed for efficient CTL priming in vivo, 2.) co-administration of the IL-2/IgG plasmid did not enhance CTL priming by the Gag p15/p26 plasmid, 3.) vaccinia vectors are useful for lentivirus-specific CTL induction in the horse, 4.) Gag-specific CTL alone are either insufficient or a more robust Gag-specific CTL response is needed to limit EIAV viremia and clinical disease, and 5.) CTL-inducing vaccines lacking envelope immunogens can result in lentiviral disease enhancement. Although the mechanisms for enhancement associated with this vaccine regimen remain to be elucidated, these results have important implications for development of lentivirus T cell vaccines. PMID:19368787

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII

PubMed Central

Fischer, Thomas H.; Herting, Jonas; Mason, Fleur E.; Hartmann, Nico; Watanabe, Saera; Nikolaev, Viacheslav O.; Sprenger, Julia U.; Fan, Peidong; Yao, Lina; Popov, Aron-Frederik; Danner, Bernhard C.; Schöndube, Friedrich; Belardinelli, Luiz; Hasenfuss, Gerd; Maier, Lars S.; Sossalla, Samuel

2015-01-01

Aims Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca2+-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca2+-leak in atrial cardiomyocytes (CMs). Methods and results In murine atrial CMs, SR-Ca2+-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca2+/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca2+-leak. The SR-Ca2+-leak induction by ATX-II was not detected when either the Na+/Ca2+ exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca2+-transient amplitude or SR-Ca2+-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca2+-transient amplitude and SR-Ca2+-load were increased, whereas PKA inhibition reduced Ca2+-transient amplitude and load and additionally slowed Ca2+ elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca2+-leak. Conclusion Late INa exerts distinct effects on Ca2+ homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca2+-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition. PMID:25990311

Crocus sativus Extract Tightens the Blood-Brain Barrier, Reduces Amyloid β Load and Related Toxicity in 5XFAD Mice.

PubMed

Batarseh, Yazan S; Bharate, Sonali S; Kumar, Vikas; Kumar, Ajay; Vishwakarma, Ram A; Bharate, Sandip B; Kaddoumi, Amal

2017-08-16

Crocus sativus, commonly known as saffron or Kesar, is used in Ayurveda and other folk medicines for various purposes as an aphrodisiac, antispasmodic, and expectorant. Previous evidence suggested that Crocus sativus is linked to improving cognitive function in Alzheimer's disease (AD) patients. The aim of this study was to in vitro and in vivo investigate the mechanism(s) by which Crocus sativus exerts its positive effect against AD. The effect of Crocus sativus extract on Aβ load and related toxicity was evaluated. In vitro results showed that Crocus sativus extract increases the tightness of a cell-based blood-brain barrier (BBB) model and enhances transport of Aβ. Further in vivo studies confirmed the effect of Crocus sativus extract (50 mg/kg/day, added to mice diet) on the BBB tightness and function that was associated with reduced Aβ load and related pathological changes in 5XFAD mice used as an AD model. Reduced Aβ load could be explained, at least in part, by Crocus sativus extract effect to enhance Aβ clearance pathways including BBB clearance, enzymatic degradation and ApoE clearance pathway. Furthermore, Crocus sativus extract upregulated synaptic proteins and reduced neuroinflammation associated with Aβ pathology in the brains of 5XFAD mice. Crocin, a major active constituent of Crocus sativus and known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo in 5XFAD mice. Crocin (10 mg/kg/day) was able to reduce Aβ load but to a lesser extent when compared to Crocus sativus extract. Collectively, findings from this study support the positive effect of Crocus sativus against AD by reducing Aβ pathological manifestations.

BSA nanoparticle loaded atorvastatin calcium--a new facet for an old drug.

PubMed

Sripriyalakshmi, S; Anjali, C H; George, Priya Doss C; Rajith, B; Ravindran, Aswathy

2014-01-01

Currently, the discovery of effective chemotherapeutic agents poses a major challenge to the field of cancer biology. The present study focuses on enhancing the therapeutic and anti cancer properties of atorvastatin calcium loaded BSA (ATV-BSA) nanoparticles in vitro. BSA-ATV nanoparticles were prepared using desolvation technique. The process parameters were optimized based on the amount of desolvating agent, stabilization conditions as well as the concentration of the cross linker. The anti cancer properties of the protein coated ATV nanoparticles were tested on MiaPaCa-2 cell lines. In vitro release behavior of the drug from the carrier suggests that about 85% of the drug gets released after 72 hrs. Our studies show that ATV-BSA nanoparticles showed specific targeting and enhanced cytotoxicity to MiaPaCa-2 cells when compared to the bare ATV. We hereby propose that the possible mechanism of cellular uptake of albumin bound ATV could be through caveolin mediated endocytosis. Hence our studies open up new facet for an existing cholesterol drug as a potent anti-cancer agent.

Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging.

PubMed

Ben-Zvi, Anat; Miller, Elizabeth A; Morimoto, Richard I

2009-09-01

Protein damage contributes prominently to cellular aging. To address whether this occurs at a specific period during aging or accumulates gradually, we monitored the biochemical, cellular, and physiological properties of folding sensors expressed in different tissues of C. elegans. We observed the age-dependent misfolding and loss of function of diverse proteins harboring temperature-sensitive missense mutations in all somatic tissues at the permissive condition. This widespread failure in proteostasis occurs rapidly at an early stage of adulthood, and coincides with a severely reduced activation of the cytoprotective heat shock response and the unfolded protein response. Enhancing stress responsive factors HSF-1 or DAF-16 suppresses misfolding of these metastable folding sensors and restores the ability of the cell to maintain a functional proteome. This suggests that a compromise in the regulation of proteostatic stress responses occurs early in adulthood and tips the balance between the load of damaged proteins and the proteostasis machinery. We propose that the collapse of proteostasis represents an early molecular event of aging that amplifies protein damage in age-associated diseases of protein conformation.

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

PubMed

Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

2017-08-01

Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese individuals.

Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium

PubMed Central

Harston, Rebecca K.; McKillop, John C.; Moschella, Phillip C.; Van Laer, An; Quinones, Lakeya S.; Baicu, Catalin F.; Balasubramanian, Sundaravadivel; Zile, Michael R.

2011-01-01

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy. PMID:21357504

Fabrication of a Soybean Bowman-Birk Inhibitor (BBI) Nanodelivery Carrier To Improve Bioavailability of Curcumin.

PubMed

Liu, Chun; Cheng, Fenfen; Yang, Xiaoquan

2017-03-22

Curcumin is a poorly water-soluble drug, and its oral bioavailability is very low. Here, a novel self-assembly nanoparticle delivery carrier has been successfully developed by using soybean Bowman-Birk inhibitor (BBI) to improve the solubility, bioaccessibility, and oral absorption of curcumin. BBI is a unique protein, which can be resistant to the pH range and proteolytic enzymes in the gastrointestinal tract (GIT), bioavailable, and not allergenic. The encapsulation efficiencies (EE) and the loading capacities (LC) of curcumin in the curcumin-loaded BBI nanoparticles (Cur-BBI-NPs, size = 90.09 nm, PDI = 0.103) were 86.17 and 10.31%, respectively. The in vitro bioaccessibility of Cur-BBI-NPs was superior to that of curcumin-loaded sodium caseinate (SC) nanoparticles (Cur-SC-NPs) (as control). Moreover, Cur-BBI-NPs significantly enhanced the bioavailability of curcumin in rats compared with Cur-SC-NPs, and the clathrin-mediated endocytosis pathway probably contributed to the favorable bioavailability of Cur-BBI-NPs, as revealed by the cellular uptake inhibition study.

Low Doses of Imatinib Induce Myelopoiesis and Enhance Host Anti-microbial Immunity

PubMed Central

Swimm, Alyson; Giver, Cynthia R.; Harris, Wayne A. C.; Laval, Julie; Napier, Brooke A.; Patel, Gopi; Crump, Ryan; Peng, Zhenghong; Bornmann, William; Pulendran, Bali; Buller, R. Mark; Weiss, David S.; Tirouvanziam, Rabindra; Waller, Edmund K.; Kalman, Daniel

2015-01-01

Imatinib mesylate (Gleevec) inhibits Abl1, c-Kit, and related protein tyrosine kinases (PTKs) and serves as a therapeutic for chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib also has efficacy against various pathogens, including pathogenic mycobacteria, where it decreases bacterial load in mice, albeit at doses below those used for treating cancer. We report that imatinib at such low doses unexpectedly induces differentiation of hematopoietic stem cells and progenitors in the bone marrow, augments myelopoiesis but not lymphopoiesis, and increases numbers of myeloid cells in blood and spleen. Whereas progenitor differentiation relies on partial inhibition of c-Kit by imatinib, lineage commitment depends upon inhibition of other PTKs. Thus, imatinib mimics “emergency hematopoiesis,” a physiological innate immune response to infection. Increasing neutrophil numbers by adoptive transfer sufficed to reduce mycobacterial load, and imatinib reduced bacterial load of Franciscella spp., which do not utilize imatinib-sensitive PTKs for pathogenesis. Thus, potentiation of the immune response by imatinib at low doses may facilitate clearance of diverse microbial pathogens. PMID:25822986

Low doses of imatinib induce myelopoiesis and enhance host anti-microbial immunity.

PubMed

Napier, Ruth J; Norris, Brian A; Swimm, Alyson; Giver, Cynthia R; Harris, Wayne A C; Laval, Julie; Napier, Brooke A; Patel, Gopi; Crump, Ryan; Peng, Zhenghong; Bornmann, William; Pulendran, Bali; Buller, R Mark; Weiss, David S; Tirouvanziam, Rabindra; Waller, Edmund K; Kalman, Daniel

2015-03-01

Imatinib mesylate (Gleevec) inhibits Abl1, c-Kit, and related protein tyrosine kinases (PTKs) and serves as a therapeutic for chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib also has efficacy against various pathogens, including pathogenic mycobacteria, where it decreases bacterial load in mice, albeit at doses below those used for treating cancer. We report that imatinib at such low doses unexpectedly induces differentiation of hematopoietic stem cells and progenitors in the bone marrow, augments myelopoiesis but not lymphopoiesis, and increases numbers of myeloid cells in blood and spleen. Whereas progenitor differentiation relies on partial inhibition of c-Kit by imatinib, lineage commitment depends upon inhibition of other PTKs. Thus, imatinib mimics "emergency hematopoiesis," a physiological innate immune response to infection. Increasing neutrophil numbers by adoptive transfer sufficed to reduce mycobacterial load, and imatinib reduced bacterial load of Franciscella spp., which do not utilize imatinib-sensitive PTKs for pathogenesis. Thus, potentiation of the immune response by imatinib at low doses may facilitate clearance of diverse microbial pathogens.

Effects of L-arginine on solubilization and purification of plant membrane proteins.

PubMed

Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

2011-11-01

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Xiaolin; Li, Qian; Pang, Liewen

Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-densitymore » lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.« less

Protein-Containing Multilayer Capsules by Templating on Mesoporous CaCO3 Particles: POST- and PRE-Loading Approaches.

PubMed

Balabushevich, Nadezhda G; Lopez de Guerenu, Anna V; Feoktistova, Natalia A; Skirtach, Andre G; Volodkin, Dmitry

2016-01-01

Encapsulation of model proteins (catalase, insulin, aprotinin) into multilayer dextran sulphate/protamin capsules by templating on CaCO3 microparticles is investigated employing: (i) PRE-loading into CaCO3 particles by adsorption or co-synthesis and (ii) POST-loading into performed capsules. Protein encapsulation is governed by both its size and electrostatic interactions with the carbonate microparticles and multilayer shell. PRE-loading enables improved encapsulation compared to POST-loading (catalase content in capsules 630 and 70 mg · g(-1)). Bioactivity of encapsulated protein is not affected by interaction with multilayers but may be reduced at slightly alkaline pH due to CaCO3 hydrolysis. This study might help to successfully encapsulate fragile bio-macromolecules into multilayer capsules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Versatile three-dimensional virus-based template for dye-sensitized solar cells with improved electron transport and light harvesting.

PubMed

Chen, Po-Yen; Dang, Xiangnan; Klug, Matthew T; Qi, Jifa; Dorval Courchesne, Noémie-Manuelle; Burpo, Fred J; Fang, Nicholas; Hammond, Paula T; Belcher, Angela M

2013-08-27

By genetically encoding affinity for inorganic materials into the capsid proteins of the M13 bacteriophage, the virus can act as a template for the synthesis of nanomaterial composites for use in various device applications. Herein, the M13 bacteriophage is employed to build a multifunctional and three-dimensional scaffold capable of improving both electron collection and light harvesting in dye-sensitized solar cells (DSSCs). This has been accomplished by binding gold nanoparticles (AuNPs) to the virus proteins and encapsulating the AuNP-virus complexes in TiO2 to produce a plasmon-enhanced and nanowire (NW)-based photoanode. The NW morphology exhibits an improved electron diffusion length compared to traditional nanoparticle-based DSSCs, and the AuNPs increase the light absorption of the dye-molecules through the phenomenon of localized surface plasmon resonance. Consequently, we report a virus-templated and plasmon-enhanced DSSC with an efficiency of 8.46%, which is achieved through optimizing both the NW morphology and the concentration of AuNPs loaded into the solar cells. In addition, we propose a theoretical model that predicts the experimentally observed trends of plasmon enhancement.

p62-enriched inclusion bodies in macrophages protect against atherosclerosis

PubMed Central

Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J.; Evans, Trent D.; Arif, Batool; Curci, John A.; Razani, Babak

2016-01-01

Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages that cannot undergo autophagy because of a deficiency of an autophagy component such as ATG5. We showed that exposure of macrophages to atherogenic lipids led to an increase in the abundance of the autophagy chaperone p62, which colocalized with polyubiquitinated proteins in cytoplasmic inclusions. p62 accumulation was increased in ATG5-null macrophages, which had large cytoplasmic ubiquitin-positive p62 inclusions. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that co-localized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. The failure of these aggregates to form was associated with increased secretion of IL-1β and enhanced macrophage apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted. PMID:26732762

The stress-induced heat shock protein 70.3 expression is regulated by a dual-component mechanism involving alternative polyadenylation and HuR.

PubMed

Kraynik, Stephen M; Gabanic, Andrew; Anthony, Sarah R; Kelley, Melissa; Paulding, Waltke R; Roessler, Anne; McGuinness, Michael; Tranter, Michael

2015-06-01

Heat shock protein 70.3 (Hsp70.3) expression increases in response to cellular stress and plays a cytoprotective role. We have previously shown that Hsp70.3 expression is controlled through coordinated post-transcriptional regulation by miRNAs and alternative polyadenylation (APA), and APA-mediated shortening of the Hsp70.3 3'-UTR facilitates increased protein expression. A stress-induced increase in Hsp70.3 mRNA and protein expression is accompanied by alternative polyadenylation (APA)-mediated truncation of the 3'UTR of the Hsp70.3 mRNA transcript. However, the role that APA plays in stress-induced expression of Hsp70.3 remains unclear. Our results show that APA-mediated truncation of the Hsp70.3 3'UTR increases protein expression through enhanced polyribosome loading. Additionally, we demonstrate that the RNA binding protein HuR, which has been previously shown to play a role in mediating APA, is necessary for heat shock mediated increase in Hsp70.3 mRNA and protein. However, it is somewhat surprising to note that HuR does not play a role in APA of the Hsp70.3 mRNA, and these two regulatory events appear to be mutually exclusive regulators of Hsp70.3 expression. These results not only provide important insight to the regulation of stress response genes following heat shock, but also contribute an enhanced understanding of how alternative polyadenylation contributes to gene regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

PubMed Central

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

2011-01-01

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

Integrated CZE-ESI-MS/MS system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate

PubMed Central

Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.

2013-01-01

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE-ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide IDs when 20 fmole was loaded. When only 1 fmole BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when low fmole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high sensitivity analysis, the integrated CZE-ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity. PMID:23510126

Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography.

PubMed

Ghose, Sanchayita; Nagrath, Deepak; Hubbard, Brian; Brooks, Clayton; Cramer, Steven M

2004-01-01

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.

Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells.

PubMed

Kasra, Mehran; Goel, Vijay; Martin, James; Wang, Shea-Tien; Choi, Woosung; Buckwalter, Joseph

2003-07-01

The pathogenesis of vibration-induced disorders of intervertebral disc at the cellular level is largely unknown. The objective of this study was to establish a method to investigate the ranges of constructive and destructive hydrostatic loading frequencies and amplitudes in preventing or inducing extracellular disc matrix degradation. Using a hydraulic chamber, normal rabbit intervertebral disc cells were tested under dynamic hydrostatic loading. Monolayer cultures of disc outer annulus cells and 3-dimensional (3-D) alginate cultures of disc nucleus pulposus cells were tested. Effects of different loading amplitudes (3-D culture, 0-3 MPa; monolayer, 0-1.7 MPa) and frequencies (1-20 Hz) on disc collagen and protein metabolism were investigated by measuring 3H-proline-labeled proteins associated with the cells in the extracellular matrix and release of 3H-proline-labeled molecules into culture medium. High frequency and high amplitude hydrostatic stress stimulated collagen synthesis in cultures of outer annulus cells whereas the lower amplitude and frequency hydrostatic stress had little effect. For the same loading duration and repetition, neither treatment significantly affected the relative amount of protein released from the cell layers, indicating that protein degradation and stability were unaffected. In the 3-D nucleus culture, higher amplitude and frequency increased synthesis rate and lowered degradation. In this case, loading amplitude had a stronger influence on cell response than that of loading frequency. Considering the ranges of loading amplitude and frequency used in this study, short-term application of high loading amplitudes and frequencies was beneficial in stimulation of protein synthesis and reduction of protein degradation.

Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility.

PubMed

Natsuga, Ken; Cipolat, Sara; Watt, Fiona M

2016-01-01

Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cell recruiting chemokine-loaded sprayable gelatin hydrogel dressings for diabetic wound healing.

PubMed

Yoon, Dong Suk; Lee, Yunki; Ryu, Hyun Aae; Jang, Yeonsue; Lee, Kyoung-Mi; Choi, Yoorim; Choi, Woo Jin; Lee, Moses; Park, Kyung Min; Park, Ki Dong; Lee, Jin Woo

2016-07-01

In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications. Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

Monte Carlo simulation of biomolecular systems with BIOMCSIM

NASA Astrophysics Data System (ADS)

Kamberaj, H.; Helms, V.

2001-12-01

A new Monte Carlo simulation program, BIOMCSIM, is presented that has been developed in particular to simulate the behaviour of biomolecular systems, leading to insights and understanding of their functions. The computational complexity in Monte Carlo simulations of high density systems, with large molecules like proteins immersed in a solvent medium, or when simulating the dynamics of water molecules in a protein cavity, is enormous. The program presented in this paper seeks to provide these desirable features putting special emphasis on simulations in grand canonical ensembles. It uses different biasing techniques to increase the convergence of simulations, and periodic load balancing in its parallel version, to maximally utilize the available computer power. In periodic systems, the long-ranged electrostatic interactions can be treated by Ewald summation. The program is modularly organized, and implemented using an ANSI C dialect, so as to enhance its modifiability. Its performance is demonstrated in benchmark applications for the proteins BPTI and Cytochrome c Oxidase.

A biomimetic approach to enhancing interfacial interactions: polydopamine-coated clay as reinforcement for epoxy resin.

PubMed

Yang, Liping; Phua, Si Lei; Teo, Jun Kai Herman; Toh, Cher Ling; Lau, Soo Khim; Ma, Jan; Lu, Xuehong

2011-08-01

A facile biomimetic method was developed to enhance the interfacial interaction in polymer-layered silicate nanocomposites. By mimicking mussel adhesive proteins, a monolayer of polydopamine was constructed on clay surface by a controllable coating method. The modified clay (D-clay) was incorporated into an epoxy resin, it is found that the strong interfacial interactions brought by the polydopamine benefits not only the dispersion of the D-clay in the epoxy but also the effective interfacial stress transfer, leading to greatly improved thermomechanical properties at very low inorganic loadings. Rheological and infrared spectroscopic studies show that the interfacial interactions between the D-clay and epoxy are dominated by the hydrogen bonds between the catechol-enriched polydopamine and the epoxy.

Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation

NASA Technical Reports Server (NTRS)

Harter, L. V.; Hruska, K. A.; Duncan, R. L.

1995-01-01

Exposure of osteosarcoma cell lines to chronic intermittent strain increases the activity of mechano-sensitive cation (SA-cat) channels. The impact of mechano-transduction on osteoblast function has not been well studied. We analyzed the expression and production of bone matrix proteins in human osteoblast-like osteosarcoma cells, OHS-4, in response to chronic intermittent mechanical strain. The OHS-4 cells exhibit type I collagen production, 1,25-Dihydroxyvitamin D-inducible osteocalcin, and mineralization of the extracellular matrix. The matrix protein message level was determined from total RNA isolated from cells exposed to 1-4 days of chronic intermittent strain. Northern analysis for type I collagen indicated that strain increased collagen message after 48 h. Immunofluorescent labeling of type I collagen demonstrated that secretion was also enhanced with mechanical strain. Osteopontin message levels were increased several-fold by the application of mechanical load in the absence of vitamin D, and the two stimuli together produced an additive effect. Osteocalcin secretion was also increased with cyclic strain. Osteocalcin levels were not detectable in vitamin D-untreated control cells. However, after 4 days of induced load, significant levels of osteocalcin were observed in the medium. With vitamin D present, osteocalcin levels were 4 times higher in the medium of strained cells compared to nonstrained controls. We conclude that mechanical strain of osteoblast-like cells is sufficient to increase the transcription and secretion of matrix proteins via mechano-transduction without hormonal induction.

Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

PubMed Central

Gong, Wenping; Wang, Pengcheng; Xiong, Xiaolu; Jiao, Jun; Yang, Xiaomei; Wen, Bohai

2015-01-01

The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii. PMID:25909586

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

PubMed

Francis, Nicola L; Hunger, Philipp M; Donius, Amalie E; Wegst, Ulrike G K; Wheatley, Margaret A

2017-01-01

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. Copyright © 2014 John Wiley & Sons, Ltd.

Low rate loading-induced convection enhances net transport into the intervertebral disc in vivo.

PubMed

Gullbrand, Sarah E; Peterson, Joshua; Mastropolo, Rosemarie; Roberts, Timothy T; Lawrence, James P; Glennon, Joseph C; DiRisio, Darryl J; Ledet, Eric H

2015-05-01

The intervertebral disc primarily relies on trans-endplate diffusion for the uptake of nutrients and the clearance of byproducts. In degenerative discs, diffusion is often diminished by endplate sclerosis and reduced proteoglycan content. Mechanical loading-induced convection has the potential to augment diffusion and enhance net transport into the disc. The ability of convection to augment disc transport is controversial and has not been demonstrated in vivo. To determine if loading-induced convection can enhance small molecule transport into the intervertebral disc in vivo. Net transport was quantified via postcontrast enhanced magnetic resonance imaging (MRI) into the discs of the New Zealand white rabbit lumbar spine subjected to in vivo cyclic low rate loading. Animals were administered the MRI contrast agent gadodiamide intravenously and subjected to in vivo low rate loading (0.5 Hz, 200 N) via a custom external loading apparatus for either 2.5, 5, 10, 15, or 20 minutes. Animals were then euthanized and the lumbar spines imaged using postcontrast enhanced MRI. The T1 constants in the nucleus, annulus, and cartilage endplates were quantified as a measure of gadodiamide transport into the loaded discs compared with the adjacent unloaded discs. Microcomputed tomography was used to quantify subchondral bone density. Low rate loading caused the rapid uptake and clearance of gadodiamide in the nucleus compared with unloaded discs, which exhibited a slower rate of uptake. Relative to unloaded discs, low rate loading caused a maximum increase in transport into the nucleus of 16.8% after 5 minutes of loading. Low rate loading increased the concentration of gadodiamide in the cartilage endplates at each time point compared with unloaded levels. Results from this study indicate that forced convection accelerated small molecule uptake and clearance in the disc induced by low rate mechanical loading. Low rate loading may, therefore, be therapeutic to the disc as it may enhance the nutrient uptake and waste product clearance. Copyright © 2015 Elsevier Inc. All rights reserved.

A rapid-acting, long-acting insulin formulation based on a phospholipid complex loaded PHBHHx nanoparticles.

PubMed

Peng, Qiang; Zhang, Zhi-Rong; Gong, Tao; Chen, Guo-Qiang; Sun, Xun

2012-02-01

The application of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for sustained and controlled delivery of hydrophilic insulin was made possible by preparing insulin phospholipid complex loaded biodegradable PHBHHx nanoparticles (INS-PLC-NPs). The INS-PLC-NPs produced by a solvent evaporation method showed a spherical shape with a mean particle size, zeta potential and entrapment efficiency of 186.2 nm, -38.4 mv and 89.73%, respectively. In vitro studies demonstrated that only 20% of insulin was released within 31 days with a burst release of 5.42% in the first 8 h. The hypoglycaemic effect in STZ induced diabetic rats lasted for more than 3 days after the subcutaneous injection of INS-PLC-NPs, which significantly prolonged the therapeutic effect compared with the administration of insulin solution. The pharmacological bioavailability (PA) of INS-PLC-NPs relative to insulin solution was over 350%, indicating that the bioavailability of insulin was significantly enhanced by INS-PLC-NPs. Therefore, the INS-PLC-NPs system is promising to serve as a long lasting insulin release formulation, by which the patient compliance can be enhanced significantly. This study also showed that phospholipid complex loaded biodegradable nanoparticles (PLC-NPs) have a great potential to be used as a sustained delivery system for hydrophilic proteins to be encapsulated in hydrophobic polymers. Copyright © 2011 Elsevier Ltd. All rights reserved.

Intra-articular TSG-6 delivery from heparin-based microparticles reduces cartilage damage in a rat model of osteoarthritis.

PubMed

Tellier, Liane E; Treviño, Elda A; Brimeyer, Alexandra L; Reece, David S; Willett, Nick J; Guldberg, Robert E; Temenoff, Johnna S

2018-05-01

As a potential treatment for osteoarthritis (OA), we have developed injectable and hydrolytically degradable heparin-based biomaterials with tunable sulfation for the intra-articular delivery of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a protein known to inhibit plasmin which may degrade extracellular matrix within OA joints. We first assessed the effect of heparin sulfation on TSG-6 anti-plasmin activity and found that while fully sulfated (Hep) and heparin desulfated at only the N position (Hep-N) significantly enhanced TSG-6 bioactivity in vitro, fully desulfated heparin (Hep-) had no effect, indicating that heparin sulfation plays a significant role in modulating TSG-6 bioactivity. Next, TSG-6 loaded, degradable 10 wt% Hep-N microparticles (MPs) were delivered via intra-articular injection into the knee at 1, 7, and 15 days following medial meniscal transection (MMT) injury in a rat model. After 21 days, cartilage thickness, volume, and attenuation were significantly increased with soluble TSG-6, indicating degenerative changes. In contrast, no significant differences were observed with TSG-6 loaded MP treatment, demonstrating that TSG-6 loaded MPs reduced cartilage damage following MMT injury. Ultimately, our results indicate that Hep-N can enhance TSG-6 anti-plasmin activity and that Hep-N-based biomaterials may be an effective method for TSG-6 delivery to treat OA.

Halloysite Clay Nanotubes for Loading and Sustained Release of Functional Compounds.

PubMed

Lvov, Yuri; Wang, Wencai; Zhang, Liqun; Fakhrullin, Rawil

2016-02-10

Halloysite is an alumosilicate tubular clay with a diameter of 50 nm, an inner lumen of 15 nm and a length of 600-900 nm. It is a natural biocompatible nanomaterial available in thousands of tons at low price, which makes it a good candidate for nanoarchitectural composites. The inner lumen of halloysite may be adjusted by etching to 20-30% of the tube volume and loading with functional agents (antioxidants, anticorrosion agents, flame-retardant agents, drugs, or proteins) allowing for formulations with sustained release tuned by the tube end-stoppers for hours and days. Clogging the tube ends in polymeric composites allows further extension of the release time. Thus, antioxidant-loaded halloysite doped into rubber enhances anti-aging properties for at least 12 months. The addition of 3-5 wt% of halloysite increases the strength of polymeric materials, and the possibility of the tube's orientation promises a gradient of properties. Halloysite nanotubes are a promising mesoporous media for catalytic nanoparticles that may be seeded on the tube surface or synthesized exclusively in the lumens, providing enhanced catalytic properties, especially at high temperatures. In vitro and in vivo studies on biological cells and worms indicate the safety of halloysite, and tests for efficient adsorption of mycotoxins in animals' stomachs are also carried out. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of different protein hydrolysate/carbohydrate mixtures on postprandial glucagon and insulin responses in healthy subjects.

PubMed

Claessens, M; Calame, W; Siemensma, A D; van Baak, M A; Saris, W H M

2009-01-01

To study the effect of four protein hydrolysates from vegetable (pea, gluten, rice and soy) and two protein hydrolysates from animal origin (whey and egg) on glucagon and insulin responses. Eight healthy normal-weight male subjects participated in this study. The study employed a repeated-measures design with Latin square randomization and single-blind trials. Protein hydrolysates used in this study (pea, rice, soy, gluten, whey and egg protein hydrolysate) consisted of 0.2 g hydrolysate per kg body weight (bw) and 0.2 g maltodextrin per kg bw and were compared to maltodextrin alone. Postprandial plasma glucose, glucagon, insulin and amino acids were determined over 2 h. All protein hydrolysates induced an enhanced insulin secretion compared to maltodextrin alone and a correspondingly low plasma glucose response. A significant difference was observed in area under the curve (AUC) for plasma glucagon between protein hydrolysates and the maltodextrin control drink (P<0.05). Gluten protein hydrolysate induced the lowest glucagon response. High amino-acid-induced glucagon response does not necessarily go together with low insulin response. Protein hydrolysate source affects AUC for glucagon more profoundly than for insulin, although the protein load used in this study seemed to be at lower level for significant physiological effects.

Low-density lipoproteins modulate endothelial cells to secrete endothelin-1 in a polarized pattern: a study using a culture model system simulating arterial intima.

PubMed

Unoki, H; Fan, J; Watanabe, T

1999-01-01

We investigated the structural and functional properties of human umbilical vein endothelial cells (HUVECs) cultured on a two-chamber culture model system using an amnion membrane. Compared to HUVECs cultured on a plastic dish, HUVECs cultured on the model system exhibited several features similar to those of in vivo vessels, including formation of the intercellular junctional devices and expression of tight junction-associated protein ZO-1 and adherence junction-associated protein alpha-catenin. Furthermore, we found that HUVECs had a property of polar secretion of endothelin-1 (ET-1). About 90% of the total amount of synthesized ET-1 was found in the lower well, designated as the basal side. When HUVECs were incubated with either native low-density lipoproteins (nLDLs) or oxidized LDLs (oxLDLs) at a concentration of 100 microgram/ml, ET-1 secretion was significantly increased, dependent on the cell side (apical vs basal) on which the nLDLs or oxLDLs were loaded. When the LDLs were loaded on the apical side, the secretion of ET-1 from HUVECs on the apical side was increased by 48% (nLDL) and 61% (oxLDL), whereas it was accompanied by a concomitant decrease of ET-1 on the basal side (45% by nLDLs and 38% by oxLDLs). When loaded on the basal side, however, ET-1 was increased by 23% (nLDLs) and 53% (oxLDLs) on the basal side, with a 26% simultaneous decrease of ET-1 on the opposite side for both nLDLs and oxLDLs. On the contrary, high-density lipoproteins (HDLs) inhibited ET-1 secretion from HUVECs on the opposite side of the well on which HDLs were loaded; there was a 57% decrease on the basal side when HDLs were loaded on the apical side, and a 46% decrease on the apical side when loaded on the basal side. These results indicate that modulation of ET-1 secretion from ECs by lipoproteins is virtually dependent on the place (apical vs basal) where these proteins are present. The finding that nLDLs and oxLDLs enhance ET-1 secretion by ECs in a polarized pattern suggests that ET-1 may be involved in pathophysiological processes such as atherogenesis.

Biological applications of zinc imidazole framework through protein encapsulation

NASA Astrophysics Data System (ADS)

Kumar, Pawan; Bansal, Vasudha; Paul, A. K.; Bharadwaj, Lalit M.; Deep, Akash; Kim, Ki-Hyun

2016-10-01

The robustness of biomolecules is always a significant challenge in the application of biostorage in biotechnology or pharmaceutical research. To learn more about biostorage in porous materials, we investigated the feasibility of using zeolite imidazolate framework (ZIF-8) with respect to protein encapsulation. Here, bovine serum albumin (BSA) was selected as a model protein for encapsulation with the synthesis of ZIF-8 using water as a media. ZIF-8 exhibited excellent protein adsorption capacity through successive adsorption of free BSA with the formation of hollow crystals. The loading of protein in ZIF-8 crystals is affected by the molecular weight due to diffusion-limited permeation inside the crystals and also by the affinity of the protein to the pendent group on the ZIF-8 surface. The polar nature of BSA not only supported adsorption on the solid surface, but also enhanced the affinity of crystal spheres through weak coordination interactions with the ZIF-8 framework. The novel approach tested in this study was therefore successful in achieving protein encapsulation with porous, biocompatible, and decomposable microcrystalline ZIF-8. The presence of both BSA and FITC-BSA in ZIF-8 was confirmed consistently by spectroscopy as well as optical and electron microscopy.

QbD-based development of α-linolenic acid potentiated nanoemulsion for targeted delivery of doxorubicin in DMBA-induced mammary gland carcinoma: in vitro and in vivo evaluation.

PubMed

Tripathi, Chandra Bhushan; Parashar, Poonam; Arya, Malti; Singh, Mahendra; Kanoujia, Jovita; Kaithwas, Gaurav; Saraf, Shubhini A

2018-05-10

Breast cancer is the most common cancer of occurrence in women and has the highest mortality incidence rate therein. The present study envisaged to develop doxorubicin (Dox) loaded folate functionalized nanoemulsion (NE) for profound therapeutic activity against mammary gland cancer. NE was prepared using pseudo-ternary phase diagrams utilizing α-linolenic acid (ALA) as lipid phase, to further enhance the anticancer potential of Dox. Box-Behnken design was employed to systematically develop the NE. Optimized NE (f-Dox-NE) was evaluated for in vitro and in vivo performance. f-Dox-NE, with globule size 55.2 ± 3.3 nm, zeta potential - 31 ± 2 mV, entrapment 92.51 ± 3.62%, drug loading 0.42 ± 0.08% and percent drug release 94.86 ± 1.87% in 72 h, was capable of reducing cell viability in MCF-7 cell lines vis-à-vis pure and marketed drug. Further, mechanistic studies in MCF-7 cell lines demonstrated that f-Dox-NE induces cellular apoptosis by reactive oxygen species generated and mitochondrial membrane mediated apoptosis. The antitumor effect was evaluated in 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary gland tumor in female Albino Wistar rats. f-Dox-NE exhibited enhanced antitumor targeting potential, therapeutic safety and efficacy vis-à-vis pure and marketed drug, as revealed by tumor volume, animal survival, weight variation, cardiotoxicity and biodistribution studies. f-Dox-NE restored the biochemical parameters viz., SOD, catalase, TBARS and protein carbonyl, towards normal levels in comparison to DMBA induced animal group. f-Dox-NE displayed downregulation of anti-apoptotic (Bcl-2 and MMP-9) proteins and upregulation of pro-apoptotic proteins (caspase-9 and BAX). The experimental results suggest that ALA augmented folate functionalized NE are able to overcome the challenges of developing safe and effective delivery system with enhanced potential for mammary gland carcinoma therapy.

Ring finger protein 39 genetic variants associate with HIV-1 plasma viral loads and its replication in cell culture.

PubMed

Lin, Ying-Ju; Chen, Chia-Yen; Jeang, Kuan-Teh; Liu, Xiang; Wang, Jen-Hsien; Hung, Chien-Hui; Tsang, Hsinyi; Lin, Ting-Hsu; Liao, Chiu-Chu; Huang, Shao-Mei; Lin, Cheng-Wen; Ho, Mao-Wang; Chien, Wen-Kuei; Chen, Jin-Hua; Ho, Tsung-Jung; Tsai, Fuu-Jen

2014-01-01

The human immunodeficiency virus (HIV-1) exploits host proteins to complete its life cycle. Genome-wide siRNA approaches suggested that host proteins affect HIV-1 replication. However, the results barely overlapped. RING finger protein 39 (RNF39) has been identified from genome-wide association studies. However, its function during HIV-1 replication remains unclear. We investigated the relationship between common RNF39 genetic variants and HIV-1 viral loads. The effect of RNF39 protein knockdown or overexpression on HIV-1 replication was then investigated in different cell lines. Two genetic variants were associated with HIV-1 viral loads. Patients with the ht1-GG/GG haplotype presented lower RNF39 expression levels and lower HIV-1 viral load. RNF39 knockdown inhibited HIV-1 expression. RNF39 protein may be involved in HIV-1 replication as observed in genetic studies on patients with HIV-1 and in in vitro cell cultures.

Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy.

PubMed

Ding, Jian; Zhang, Chunling; Gao, Minjie; Hou, Guoli; Liang, Kexue; Li, Chunhua; Ni, Jianping; Li, Zhen; Shi, Zhongping

2014-05-10

Porcine circovirus Cap protein production by P. pastoris with strong AOX promoter suffered with the problems with traditional pure methanol induction: (1) inefficient methanol metabolism; (2) extensive oxygen supply load; (3) difficulty in stable DO control; (4) low protein titer. In this study, based on the difference of DO change patterns in response to methanol and sorbitol additions, a novel fuzzy control system was proposed to automatically regulate the co-feeding rates of methanol and sorbitol for efficient Cap protein induction. With aid of the proposed control system when setting DO control level at 10%, overall fermentation performance was significantly improved: (1) DO could be stably controlled under mild aeration condition; (2) methanol consumption rate could be restricted at moderate level and the major enzymes involved with methanol metabolism were largely activated; (3) Cap protein concentration reached a highest level of 198mg/L, which was about 64% increase over the best one using the pure methanol induction strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy.

PubMed

Jing, Hui; Cheng, Wen; Li, Shouqiang; Wu, Bolin; Leng, Xiaoping; Xu, Shouping; Tian, Jiawei

2016-10-01

The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

PubMed

Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara Dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro Dos Santos; Behrens, Maria Dutra; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

2016-08-01

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

PubMed Central

Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro dos Santos; Behrens, Maria Dutra

2016-01-01

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography–mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment. PMID:27579922

PLGA-soya lecithin based micelles for enhanced delivery of methotrexate: Cellular uptake, cytotoxic and pharmacokinetic evidences.

PubMed

Singh, Anupama; Thotakura, Nagarani; Kumar, Rajendra; Singh, Bhupinder; Sharma, Gajanand; Katare, Om Prakash; Raza, Kaisar

2017-02-01

Biocompatible and biodegradable polymers like PLGA have revolutionized the drug delivery approaches. However, poor drug loading and substantially high lipophilicity, pave a path for further tailing of this promising agent. In this regard, PLGA was feathered with biocompatible phospholipid and polymeric micelles were developed for delivery of Methotrexate (MTX) to cancer cells. The nanocarriers (114.6nm±5.5nm) enhanced the cytotoxicity of MTX by 2.13 folds on MDA-MB-231 cells. Confocal laser scanning microscopy confirmed the increased intracellular delivery. The carrier decreased the protein binding potential and enhanced the bioavailable fraction of MTX. Pharmacokinetic studies vouched substantial enhancement in AUC and bioresidence time, promising an ideal carrier to effectively deliver the drug to the site of action. The developed nanocarriers offer potential to deliver the drug in the interiors of cancer cells in an effective manner for improved therapeutic action. Copyright © 2016 Elsevier B.V. All rights reserved.

Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins.

PubMed

Dyawanapelly, Sathish; Koli, Uday; Dharamdasani, Vimisha; Jain, Ratnesh; Dandekar, Prajakta

2016-08-01

The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (D, L-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes.

Enhancement of fermentable sugar yield by competitive adsorption of non-enzymatic substances from yeast and cellulase on lignin

PubMed Central

2014-01-01

Background Enhancement of enzymatic digestibility by some supplementations could reduce enzyme loading and cost, which is still too high to realize economical production of lignocellulosic biofuels. A recent study indicates that yeast hydrolysates (YH) have improved the efficiency of cellulases on digestibility of furfural residues (FR). In the current work, the components of YH were separated by centrifugation and size exclusion chromatography and finally characterized in order to better understand this positive effect. Results A 60.8% of nitrogen of yeast cells was remained in the slurry (YHS) after hydrothermal treatment. In the supernatant of YH (YHL), substances of high molecular weight were identified as proteins and other UV-absorbing compounds, which showed close molecular weight to components of cellulases. Those substances attributed to a synergetic positive effect on enzymatic hydrolysis of FR. The fraction of YHL ranged from 1.19 to 2.19 mL (elution volume) contained over 50% of proteins in YHL and had the best performance in stimulating the release of glucose. Experiment results proved the adsorption of proteins in YHL on lignin. Conclusions Supplementation of cellulases with YH enhances enzymatic digestibility of FR mainly by a competitive adsorption of non-enzymatic substances on lignin. The molecular weight of these substances has a significant impact on their performance. Different strategies can be used for a good utilization of yeast cells in terms of biorefinery concept. PMID:24650152

Dual Targeting of Amyloid-beta Clearance and Neuroinflammation as a Novel Therapeutic Approach against Alzheimer's Disease

NASA Astrophysics Data System (ADS)

Batarseh, Yazan S.

Amyloid-beta (Abeta) cascade hypothesis suggests that Alzheimer's disease (AD) is related to an imbalance between the production and clearance of Abeta peptide. Sporadic AD has been related to faulty clearance of Abeta. Accumulation of Abeta oligomers (Abetao) has been linked to several downstream toxic effects including neuroinflammation, synaptic loss, and cellular death. Abeta transport across the blood-brain barrier (BBB) is one of the primary pathways for reducing Abeta load in the brain, which work hand in hand with other parenchymal mechanisms to reduce Abeta levels including intra and extracellular degradation by a family of Abeta degrading enzymes. Established AD drugs, such as the cholinesterase inhibitor donepezil, have been reported to have several additional non-cholinergic effects that alter Abeta pathology; reduce Abeta load, anti-inflammatory response, and attenuate synaptic loss. However, their limited effect only lead to minor improvements in AD symptoms without improving the prognosis of the disease. The lack of effective medical treatment for AD led to several studies focusing on establishing new therapeutic approaches to reduce Abeta pathology. We aimed to identify and characterize natural products that are capable of enhancing the BBB clearance of Abeta in addition to reducing neuroinflammation. Our first project was to investigate the role of oleocanthal (one of the active ingredients in extra-virgin olive oil; EVOO) on attenuating Abeta toxic effects on neurons and astrocytes. We developed Abeta oligomers (Abetao) induced inflammatory environment by exposing neurons and astrocytes to accumulative doses of Abetao to investigate oleocanthal effect on modulating Abetao pathological changes in neurons and astrocytes. Our findings demonstrated oleocanthal prevented Abetao-induced synaptic proteins, SNAP-25 and PSD-95, down-regulation in neurons, attenuated Abetao-induced inflammation, and restored glutamine transporter (GLT1) and glucose transporter (GLUT1) expressions in astrocytes. Results from this study support the protective effect of the EVOO-derived phenolic secoiridoid oleocanthal against AD pathology. Next, we evaluated the role of EVOO in enhancing donepezil's effect on increasing Abeta clearance and reducing neuroinflammation in AD transgenic model, namely 5XFAD mice. The long-term consumption of EVOO in combination with donepezil is expected to enhance and expand donepezil protective mechanisms against Abeta pathology. EVOO consumption in combination with donepezil treatment significantly reduced Abeta load and related pathology; EVOO consumption with donepezil up-regulated synaptic proteins, enhanced BBB tightness and reduced neuroinflammation associated with Abeta pathology. Long-term consumption of EVOO significantly reduced Abeta pathological manifestations in addition to enhancing and expanding donepezil protective mechanisms against Abeta pathology when given concomitantly. Therefore, EVOO consumption as a medical food combined with donepezil offers an effective therapeutic approach by enhancing the non-cholinergic mechanisms of donepezil and by providing additional mechanisms to attenuate Abeta related pathology in AD patients. In the third project, the effect of Crocus sativus extract on Abeta clearance across the BBB and related pathology were evaluated in vitro and in vivo in wild-type and AD transgenic models. Available studies reported Crocus sativus exerts a positive effect against AD, however, the mechanism(s) for such effect is unknown. Therefore, here, we investigated its effect on enhancing Abeta clearance and reducing neuroinflammation. Findings from in vitro studies demonstrated that Crocus sativus extract increased the tightness and enhanced Abeta transport in our cell-based BBB model. Followed in vivo studies confirmed the effect of Crocus sativus extract on the BBB integrity and function that was associated with reduced Abeta load and related pathology in 5XFAD mice. Furthermore, Crocus sativus extract up-regulated synaptic proteins and reduced neuroinflammation associated with Abeta pathology in the brains of 5XFAD mice. Crocin, one of the major active compounds in Crocus sativus, known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo. Crocin was able to reduce Abeta load and related pathologies but to a lesser extent when compared to Crocus sativus extract, which could be explained, at least in part, by the lack of crocin's ability in enhancing Abeta clearance and reducing neuroinflammation. Findings from this project support the positive effect of Crocus sativus against AD by reducing Abeta pathological manifestations. In conclusion, in this work, the therapeutics potential of oleocanthal, EVOO, and Crocus sativus extracts was in vitro and in vivo evaluated for their effect on Abeta clearance, BBB integrity and function, neuroprotective and neuroinflammation. Oleocanthal, EVOO, and Crocus sativus extract enhanced the clearance of Abeta by inducing its transport across the BBB and enhancing its enzymatic degradation and reduced neuroinflammation, which collectively led to Abeta brain levels reduction associated with inflammation reduction and neuroprotection. Therefore, we suggest that natural products such as EVOO, oleocanthal, and Crocus sativus may have a high potential therapeutic role against AD pathology.

Performance of homeostasis model assessment and serum high-sensitivity C-reactive protein for prediction of isolated post-load hyperglycaemia.

PubMed

Lai, Y-C; Li, H-Y; Hung, C-S; Lin, M-S; Shih, S-R; Ma, W-Y; Hua, C-H; Chuang, L-M; Sung, F-C; Wei, J-N

2013-03-01

To evaluate whether homeostasis model assessment and high-sensitivity C-reactive protein improve the prediction of isolated post-load hyperglycaemia. The subjects were 1458 adults without self-reported diabetes recruited between 2006 and 2010. Isolated post-load hyperglycaemia was defined as fasting plasma glucose < 7 mmol/l and 2-h post-load plasma glucose ≥ 11.1 mmol/l. Risk scores of isolated post-load hyperglycaemia were constructed by multivariate logistic regression. An independent group (n = 154) was enrolled from 2010 to 2011 to validate the models' performance. One hundred and twenty-three subjects (8.28%) were newly diagnosed as having diabetes mellitus. Among those with undiagnosed diabetes, 64 subjects (52%) had isolated post-load hyperglycaemia. Subjects with isolated post-load hyperglycaemia were older, more centrally obese and had higher blood pressure, HbA(1c), fasting plasma glucose, triglycerides, LDL cholesterol, high-sensitivity C-reactive protein and homeostasis model assessment of insulin resistance and lower homeostasis model assessment of β-cell function than those without diabetes. The risk scores included age, gender, BMI, homeostasis model assessment, high-sensitivity C-reactive protein and HbA(1c). The full model had high sensitivity (84%) and specificity (87%) and area under the receiver operating characteristic curve (0.91), with a cut-off point of 23.81; validation in an independent data set showed 88% sensitivity, 77% specificity and an area under curve of 0.89. Over half of those with undiagnosed diabetes had isolated post-load hyperglycaemia. Homeostasis model assessment and high-sensitivity C-reactive protein are useful to identify subjects with isolated post-load hyperglycaemia, with improved performance over fasting plasma glucose or HbA(1c) alone. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.

Structural stability and sustained release of protein from a multilayer nanofiber/nanoparticle composite.

PubMed

Vakilian, Saeid; Mashayekhan, Shohreh; Shabani, Iman; Khorashadizadeh, Mohsen; Fallah, Ali; Soleimani, Masoud

2015-04-01

The cellular microenvironment can be engineered through the utilization of various nano-patterns and matrix-loaded bioactive molecules. In this study, a multilayer system of electrospun scaffold containing chitosan nanoparticles was introduced to overcome the common problems of instability and burst release of proteins from nanofibrous scaffolds. Bovine serum albumin (BSA)-loaded chitosan nanoparticles was fabricated based on ionic gelation interaction between chitosan and sodium tripolyphosphate. Suspension electrospinning was employed to fabricate poly-ɛ-caprolacton (PCL) containing protein-loaded chitosan nanoparticles with a core-shell structure. To obtain the desired scaffold mechanical properties with enough elasticity for expansion and contraction, a hybrid mono and multilayer electrospun scaffold was fabricated using PCL containing protein-loaded chitosan nanoparticles and poly-L-lactic acid (PLLA). According to the BSA release profile, the multi-layered structure of nanofibers with two barrier layers provided a programmable release pattern of the loaded protein. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectra results showed that the electrospinning process had no significant effect on the primary and secondary structure of the protein. The results indicated a desirable biocompatibility and mechanical cues of the multilayer nanofibrous scaffolds supporting structural stability and controlled release of the protein, which can offer diverse applications in hollow organ tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Myocardial reloading after extracorporeal membrane oxygenation alters substrate metabolism while promoting protein synthesis.

PubMed

Kajimoto, Masaki; O'Kelly Priddy, Colleen M; Ledee, Dolena R; Xu, Chun; Isern, Nancy; Olson, Aaron K; Des Rosiers, Christine; Portman, Michael A

2013-08-19

Extracorporeal membrane oxygenation (ECMO) unloads the heart, providing a bridge to recovery in children after myocardial stunning. ECMO also induces stress which can adversely affect the ability to reload or wean the heart from the circuit. Metabolic impairments induced by altered loading and/or stress conditions may impact weaning. However, cardiac substrate and amino acid requirements upon weaning are unknown. We assessed the hypothesis that ventricular reloading with ECMO modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Sixteen immature piglets (7.8 to 15.6 kg) were separated into 2 groups based on ventricular loading status: 8-hour ECMO (UNLOAD) and postwean from ECMO (RELOAD). We infused into the coronary artery [2-(13)C]-pyruvate as an oxidative substrate and [(13)C6]-L-leucine as an indicator for amino acid oxidation and protein synthesis. Upon RELOAD, each functional parameter, which were decreased substantially by ECMO, recovered to near-baseline level with the exclusion of minimum dP/dt. Accordingly, myocardial oxygen consumption was also increased, indicating that overall mitochondrial metabolism was reestablished. At the metabolic level, when compared to UNLOAD, RELOAD altered the contribution of various substrates/pathways to tissue pyruvate formation, favoring exogenous pyruvate versus glycolysis, and acetyl-CoA formation, shifting away from pyruvate decarboxylation to endogenous substrate, presumably fatty acids. Furthermore, there was also a significant increase of tissue concentrations for all CAC intermediates (≈80%), suggesting enhanced anaplerosis, and of fractional protein synthesis rates (>70%). RELOAD alters both cytosolic and mitochondrial energy substrate metabolism, while favoring leucine incorporation into protein synthesis rather than oxidation in the CAC. Improved understanding of factors governing these metabolic perturbations may serve as a basis for interventions and thereby improve success rate from weaning from ECMO.

A SENSITIVE FLUORESCENCE-BASED ASSAY FOR MONITORING GM2 GANGLIOSIDE HYDROLYSIS IN LIVE PATIENT CELLS AND THEIR LYSATES

PubMed Central

Tropak, Michael B.; Bukovac, Scott W.; Rigat, Brigitte A.; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J.

2010-01-01

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is anattractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are alsoinhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Herewe demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the β-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant β-hexosaminidase A and substrate-hydrolysis as compared to mock treated cells. PMID:19917668

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

PubMed

Tropak, Michael B; Bukovac, Scott W; Rigat, Brigitte A; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J

2010-03-01

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

Combination of bone morphogenetic protein-2 plasmid DNA with chemokine CXCL12 creates an additive effect on bone formation onset and volume.

PubMed

Wegman, F; Poldervaart, M T; van der Helm, Y J; Oner, F C; Dhert, W J; Alblas, J

2015-07-27

Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.

Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

PubMed

Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

2016-01-01

Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Cargo and Carrier Effects of Rapamycin-Loaded Perfluorocarbon Nanoparticles

NASA Astrophysics Data System (ADS)

Bibee, Kristin Page

Nanoparticle-based drug delivery has been championed as a means to increase local delivery of therapeutics while decreasing systemic drug exposure. By targeting the particles, and therefore the drugs, to diseased cells of interest, healthy cells will be spared and side effects avoided. This delivery mechanism would be particularly useful for drugs that interfere with cell growth and proliferation pathways, as blocking proliferation in normal cells leads to significant patient morbidity. Rapamycin is a macrolide and a known inhibitor of mTORC1, a protein complex that plays a crucial role in protein translation and cell growth. This work demonstrates the effects of rapamycin complexed with a nanoparticle carrier on two distinct pathologies: a new triple negative breast cancer cell line and a conventional mouse model of muscular dystrophy (mdx). Rapamycin is able to alter mitochondrial function and thus metabolism in both free and nanoparticle-delivered form without killing the cells. Although nanoparticles are considered to be a benign carrier, this work shows that perfluorocarbon nanoparticles are able to induce autophagy in vitro. The benefits of autophagy induction in cancer cells is cell and stage specific, but has been reported to be useful for radiosensitization of triple negative breast cancers. Additionally, the particles are shown to induce autophagy in the mdx model of Duchenne Muscular Dystrophy and, when loaded with rapamycin, dramatically improve strength even in older animals with muscular dystrophy. Overall, this work enhances our understanding of the cellular effects of perfluorocarbon nanoparticles in two different disease models and enhances prospects for clinical translation of nanoparticle-based drug delivery.

Cell type-selective disease-association of genes under high regulatory load

PubMed Central

Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

2015-01-01

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

Emulsifying properties and oil/water (O/W) interface adsorption behavior of heated soy proteins: effects of heating concentration, homogenizer rotating speed, and salt addition level.

PubMed

Cui, Zhumei; Chen, Yeming; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei

2014-02-19

The adsorption of heat-denatured soy proteins at the oil/water (O/W) interface during emulsification was studied. Protein samples were prepared by heating protein solutions at concentrations of 1-5% (w/v) and were then diluted to 0.3% (w/v). The results showed that soy proteins that had been heated at higher concentrations generated smaller droplet size of emulsion. Increase in homogenizer rotating speed resulted in higher protein adsorption percentages and lower surface loads at the O/W interface. Surface loads for both unheated and heated soy proteins were linearly correlated with the unadsorbed proteins' equilibrium concentration at various rotating speeds. With the rise in NaCl addition level, protein adsorption percentage and surface loads of emulsions increased, whereas lower droplet sizes were obtained at the ionic strength of 0.1 M. The aggregates and non-aggregates displayed different adsorption behaviors when rotating speed or NaCl concentration was varied.

Physical Weight Loading Induces Expression of Tryptophan Hydroxylase 2 in the Brain Stem

PubMed Central

Shim, Joon W.; Dodge, Todd R.; Hammond, Max A.; Wallace, Joseph M.; Zhou, Feng C.; Yokota, Hiroki

2014-01-01

Sustaining brain serotonin is essential in mental health. Physical activities can attenuate mental problems by enhancing serotonin signaling. However, such activity is not always possible in disabled individuals or patients with dementia. Knee loading, a form of physical activity, has been found to mimic effects of voluntary exercise. Focusing on serotonergic signaling, we addressed a question: Does local mechanical loading to the skeleton elevate expression of tryptophan hydroxylase 2 (tph2) that is a rate-limiting enzyme for brain serotonin? A 5 min knee loading was applied to mice using 1 N force at 5 Hz for 1,500 cycles. A 5-min treadmill running was used as an exercise (positive) control, and a 90-min tail suspension was used as a stress (negative) control. Expression of tph2 was determined 30 min – 2 h in three brain regions ––frontal cortex (FC), ventromedial hypothalamus (VMH), and brain stem (BS). We demonstrated for the first time that knee loading and treadmill exercise upregulated the mRNA level of tph2 in the BS, while tail suspension downregulated it. The protein level of tph2 in the BS was also upregulated by knee loading and downregulated by tail suspension. Furthermore, the downregulation of tph2 mRNA by tail suspension can be partially suppressed by pre-application of knee loading. The expression of tph2 in the FC and VMH was not significantly altered with knee loading. In this study we provided evidence that peripheral mechanical loading can activate central tph2 expression, suggesting that physical cues may mediate tph2-cathalyzed serotonergic signaling in the brain. PMID:24416346

The application of halloysite tubule nanoclay in drug delivery.

PubMed

Lvov, Yuri M; DeVilliers, Melgardt M; Fakhrullin, Rawil F

2016-07-01

Natural and biocompatible clay nanotubes are among the best inorganic materials for drug nanoformulations. These halloysite tubes with SiO2 on the outermost surface have diameter of ca. 50 nm, length around 1 micrometer and may be loaded with drugs at 10-30 wt. %. Narrow tube openings allow for controllable sustained drug release for hours, days or even weeks. Physical-chemical properties of these nanotubes are described followed by examples of drug-loading capabilities, release characteristics, and control of duration of release through the end tube capping with polymers. Development of halloysite-polymer composites such as tissue scaffolds and bone cement/dentist resin formulations with enhanced mechanical properties and extension of the drug release to 2-3 weeks are described. Examples of the compression properties of halloysite in tablets and capsules are also shown. We expect that clay nanotubes will be used primarily for non-injectable drug formulations, such as topical and oral dosage forms, cosmetics, as well as for composite materials with enhanced therapeutic effects. These include tissue scaffolds, bone cement and dentist resins with sustained release of antimicrobial and cell growth-promoting medicines (including proteins and DNA) as well as other formulations such as compounds for antiseptic treatment of hospitals.

A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mondal, Tapan Kumar; Emeny, Rebecca T.; Gao, Donghong

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2 h, host defenses against bacterial growth are suppressed 2–3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidationmore » (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes. - Highlights: • Cold-restraint (physical/psychological stress) induces early oxidative stress. • The oxidative stress relates to catecholamine signaling beta-adrenoceptors. • Physical/psychological stress combines infection enhancing inflammation. • Endoplasmic reticulum stress interferes with host defenses and autophagy.« less

Enhancement of Buckling Load with the Use of Active Materials

NASA Technical Reports Server (NTRS)

Yuan, F. G.

2002-01-01

In this paper, active buckling control of a beam using piezoelectric materials is investigated. Under small deformation, mathematical models are developed to describe the behavior of the beams subjected to an axial compressive load with geometric imperfections and load eccentricities under piezoelectric force. Two types of supports, simply supported and clamped, of the beam with a partially bonded piezoelectric actuator are used to illustrate the concept. For the beam with load eccentricities and initial geometric imperfections, the load- carrying capacity can be significantly enhanced by counteracting moments from the piezoelectric actuator. For the single piezoelectric actuator, using static feedback closed-loop control, the first buckling load can be eliminated. In the case of initially straight beams, analytical solutions of the enhanced first critical buckling load due to the increase of bending stiffness by piezoelectric actuators are derived based on linearized buckling analysis.

Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

PubMed

Tellier, Liane E; Miller, Tobias; McDevitt, Todd C; Temenoff, Johnna S

2015-10-28

Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

Optimization of novel pentablock copolymer based composite formulation for sustained delivery of peptide/protein in the treatment of ocular diseases

PubMed Central

Patel, Sulabh P.; Vaishya, Ravi; Patel, Ashaben; Agrahari, Vibhuti; Pal, Dhananjay; Mitra, Ashim K.

2016-01-01

This manuscript is focused on the development of pentablock (PB) copolymer based sustained release formulation for the treatment of posterior segment ocular diseases. We have successfully synthesized biodegradable and biocompatible PB copolymers for the preparation of nanoparticles (NPs) and thermosensitive gel. Achieving high drug loading with hydrophilic biotherapeutics (peptides /proteins) is a challenging task. Moreover, small intravitreal injection volume (≤100 μL) requires high loading to develop a long term (6 months) sustained release formulation. We have successfully investigated various formulation parameters to achieve maximum peptide/protein (octreotide, insulin, lysozyme, IgG-Fab, IgG, and catalase) loading in PB NPs. Improvement in drug loading can facilitate delivery of larger doses of therapeutic proteins via limited injection volume. A composite formulation comprised of NPs in gel system exhibited sustained release (without burst effect) of peptides and proteins, may serve as a platform technology for the treatment of posterior segment ocular diseases. PMID:26964498

Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis

NASA Astrophysics Data System (ADS)

Liu, Rui; Yu, Xiwei; Su, Chang; Shi, Yijie; Zhao, Liang

2017-06-01

Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate's reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.

5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.

PubMed

Parmar, Rubina; Willoughby, Jennifer L S; Liu, Jingxuan; Foster, Donald J; Brigham, Benjamin; Theile, Christopher S; Charisse, Klaus; Akinc, Akin; Guidry, Erin; Pei, Yi; Strapps, Walter; Cancilla, Mark; Stanton, Matthew G; Rajeev, Kallanthottathil G; Sepp-Lorenzino, Laura; Manoharan, Muthiah; Meyers, Rachel; Maier, Martin A; Jadhav, Vasant

2016-06-02

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

3D-printed dimethyloxallyl glycine delivery scaffolds to improve angiogenesis and osteogenesis.

PubMed

Min, Zhu; Shichang, Zhao; Chen, Xin; Yufang, Zhu; Changqing, Zhang

2015-08-01

Angiogenesis-osteogenesis coupling processes are vital in bone tissue engineering. Normal biomaterials implanted in bone defects have issues in the sufficient formation of blood vessels, especially in the central part. Single delivery of vascular endothelial growth factors (VEGF) to foci in previous studies did not show satisfactory results due to low loading doses, a short protein half-life and low efficiency. Development of a hypoxia-mimicking microenvironment for cells by local prolyl-4-hydroxylase inhibitor release, which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression, is an alternative method. The aim of this study was to design a dimethyloxallyl glycine (DMOG) delivering scaffold composed of mesoporous bioactive glasses and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers (MPHS scaffolds), so as to investigate whether the sustained release of DMOG promotes local angiogenesis and bone healing. The morphology and microstructure of composite scaffolds were characterized. The DMOG release patterns from scaffolds loaded with different DMOG dosages were evaluated, and the effects of DMOG delivery on human bone marrow stromal cell (hBMSC) adhesion, viability, proliferation, osteogenic differentiation and angiogenic-relative gene expressions with scaffolds were also investigated. In vivo studies were carried out to observe vascular formations and new bone ingrowth with DMOG-loaded scaffolds. The results showed that DMOG could be released in a sustained manner over 4 weeks from MPHS scaffolds and obviously enhance the angiogenesis and osteogenesis in the defects. Microfil perfusion showed a significantly increased formation of vessels in the defects with DMOG delivery. Furthermore, micro-CT imaging and fluorescence labeling indicated larger areas of bone formation for DMOG-loaded scaffolds. It is concluded that MPHS-DMOG scaffolds are promising for enhancing bone healing of osseous defects.

Incorporating Phage Therapy into WPI Dip Coatings for Applications on Fresh Whole and Cut Fruit and Vegetable Surfaces.

PubMed

Vonasek, Erica L; Choi, Angela H; Sanchez, Juan; Nitin, Nitin

2018-06-15

There is a significant unmet need to develop antimicrobial solutions to reduce the risk of contamination in fresh produce. Bacteriophages have been proposed as a potential approach for controlling foodborne pathogens. This study evaluated the combination of edible dip coatings with T7 bacteriophages on whole and cut produce. The evaluation includes an assessment of phage loading, phage storage stability, antimicrobial activity, and phage stability during simulated gastric digestion on sliced cucumbers, sliced apples, and whole cherry tomatoes. In this evaluation, phages coated on fresh produce using edible whey protein isolate (WPI) were compared with phages coated from an aqueous suspension (control coating). The results demonstrated that WPI coatings load more phages than the control and enhanced phage stability during cold storage (4 °C) for cut apples and whole cherry tomatoes. Phage stability decreased by 1 to 3 log(PFU) in a simulated gastric environment. Phage antimicrobial activity against Escherichia coli BL21 decreased 2 to 4 log(CFU) of bacteria on cut apples and whole cherry tomatoes, while no significant bacterial reduction was observed for sliced cucumbers. Overall, the results show that WPI dip coating provides phage loading, stability, and antimicrobial activity to produce surfaces compared to the control coating, and thus may be considered an effective approach for extending phage therapy on fresh produce. The practical application is to prevent bacterial cross contamination of fresh produce by using a combination of edible coating with bacteriophages. The results demonstrate enhanced loading and stability of phages on fresh produce when used in combination with an edible coating. © 2018 Institute of Food Technologists®.

The synergistic effect of VEGF and biomorphic silicon carbides topography on in vivo angiogenesis and human bone marrow derived mesenchymal stem cell differentiation.

PubMed

Díaz-Rodríguez, P; Gómez-Amoza, J L; Landin, M

2015-08-04

Topographical features of biomaterials are able to modulate cell attachment, spreading and differentiation. The addition of growth factors to implantable biomaterials can modify these cellular responses, enhancing their therapeutic potential. The aim of this research is to establish the influence of biomorphic silicon carbide ceramics (bioSiCs) surface topography on the proliferation and osteoblastic differentiation of mesenchymal stem cells and the potential synergistic effect of the ceramic porous structure together with vascular endothelial growth factor loading (VEGF) on the surface mediated osteoblastic differentiation. Three porous bioSiCs with important differences in their microstructure were obtained from different natural precursors. Samples loaded with or without VEGF through ionic interactions were cultured with human umbilical vein endothelial cells (HUVEC) or bone marrow derived mesenchymal stem cells (hMSCs). Cell behaviour and protein activity with regard to bioSiC porous structure and surface properties were analysed. An in vivo model (Chick Chorioallantoic Membrane; CAM) was used to assess the capability of the VEGF loaded systems to promote angiogenesis. Experimental data show that loaded systems were able to control the release of VEGF for up to 15 d ensuring the activity of the protein, increasing the proliferation of HUVECs and the formation of new blood vessels in the CAM. It was found that the selection of bioSiCs with a higher pore size promoted a higher concentration of osteoblastic differentiation markers of MSCs cultured on the surface of bioSiCs. Furthermore, the addition of VEGF to the systems was able to promote a faster osteoblastic differentiation according to the qPCR results, suggesting a synergy between both the surface properties and the controlled release of the growth factor. The VEGF loaded sapelli bioSiC was found to be the most promising material for bone tissue engineering applications.

Silica-coated Gd(DOTA)-loaded protein nanoparticles enable magnetic resonance imaging of macrophages

PubMed Central

Bruckman, Michael A.; Randolph, Lauren N.; Gulati, Neetu M.; Stewart, Phoebe L.; Steinmetz, Nicole F.

2015-01-01

The molecular imaging of in vivo targets allows non-invasive disease diagnosis. Nanoparticles offer a promising platform for molecular imaging because they can deliver large payloads of imaging reagents to the site of disease. Magnetic resonance imaging (MRI) is often preferred for clinical diagnosis because it uses non-ionizing radiation and offers both high spatial resolution and excellent penetration. We have explored the use of plant viruses as the basis of for MRI contrast reagents, specifically Tobacco mosaic virus (TMV), which can assemble to form either stiff rods or spheres. We loaded TMV particles with paramagnetic Gd ions, increasing the ionic relaxivity compared to free Gd ions. The loaded TMV particles were then coated with silica maintaining high relaxivities. Interestingly, we found that when Gd(DOTA) was loaded into the interior channel of TMV and the exterior was coated with silica, the T1 relaxivities increased by three-fold from 10.9 mM−1 s−1 to 29.7 mM−1s−1 at 60 MHz compared to uncoated Gd-loaded TMV. To test the performance of the contrast agents in a biological setting, we focused on interactions with macrophages because the active or passive targeting of immune cells is a popular strategy to investigate the cellular components involved in disease progression associated with inflammation. In vitro assays and phantom MRI experiments indicate efficient targeting and imaging of macrophages, enhanced contrast-to-noise ratio was observed by shape-engineering (SNP > TMV) and silica-coating (Si-TMV/SNP > TMV/SNP). Because plant viruses are in the food chain, antibodies may be prevalent in the population. Therefore we investigated whether the silica-coating could prevent antibody recognition; indeed our data indicate that mineralization can be used as a stealth coating option to reduce clearance. Therefore, we conclude that the silica-coated protein-based contrast agent may provide an interesting candidate material for further investigation for in vivo delineation of disease through macrophage imaging. PMID:26659591

Load controller and method to enhance effective capacity of a photovoltaic power supply using a dynamically determined expected peak loading

DOEpatents

Perez, Richard

2005-05-03

A load controller and method are provided for maximizing effective capacity of a non-controllable, renewable power supply coupled to a variable electrical load also coupled to a conventional power grid. Effective capacity is enhanced by monitoring power output of the renewable supply and loading, and comparing the loading against the power output and a load adjustment threshold determined from an expected peak loading. A value for a load adjustment parameter is calculated by subtracting the renewable supply output and the load adjustment parameter from the current load. This value is then employed to control the variable load in an amount proportional to the value of the load control parameter when the parameter is within a predefined range. By so controlling the load, the effective capacity of the non-controllable, renewable power supply is increased without any attempt at operational feedback control of the renewable supply.

FPGA-based protein sequence alignment : A review

NASA Astrophysics Data System (ADS)

Isa, Mohd. Nazrin Md.; Muhsen, Ku Noor Dhaniah Ku; Saiful Nurdin, Dayana; Ahmad, Muhammad Imran; Anuar Zainol Murad, Sohiful; Nizam Mohyar, Shaiful; Harun, Azizi; Hussin, Razaidi

2017-11-01

Sequence alignment have been optimized using several techniques in order to accelerate the computation time to obtain the optimal score by implementing DP-based algorithm into hardware such as FPGA-based platform. During hardware implementation, there will be performance challenges such as the frequent memory access and highly data dependent in computation process. Therefore, investigation in processing element (PE) configuration where involves more on memory access in load or access the data (substitution matrix, query sequence character) and the PE configuration time will be the main focus in this paper. There are various approaches to enhance the PE configuration performance that have been done in previous works such as by using serial configuration chain and parallel configuration chain i.e. the configuration data will be loaded into each PEs sequentially and simultaneously respectively. Some researchers have proven that the performance using parallel configuration chain has optimized both the configuration time and area.

Proteomic determination of widespread detergent-insolubility including Abeta but not tau early in the pathogenesis of Alzheimer's disease.

PubMed

Woltjer, Randall L; Cimino, P J; Boutté, Angela M; Schantz, Aimee M; Montine, Kathleen S; Larson, Eric B; Bird, Thomas; Quinn, Joseph F; Zhang, Jing; Montine, Thomas J

2005-11-01

Biochemical characterization of the major detergent-insoluble proteins that comprise hallmark histopathologic lesions initiated the molecular era of Alzheimer's disease (AD) research. Here, we reinvestigated detergent-insoluble proteins in AD using modern proteomic techniques. Using liquid chromatography (LC)-mass spectrometry (MS)-MS-based proteomics, we robustly identified 125 proteins in the detergent-insoluble fraction of late-onset AD (LOAD) temporal cortex that included several proteins critical to Abeta production, components of synaptic scaffolding, and products of genes linked to an increased risk of LOAD; we verified 15 of 15 of these proteins by Western blot. Following multiple analyses, we estimated that these represent ~80% of detergent-insoluble proteins in LOAD detectable by our method. Abeta, tau, and 7 of 8 other newly identified detergent-insoluble proteins were disproportionately increased in temporal cortex from patients with LOAD and AD derived from mutations in PSEN1 and PSEN2; all of these except tau were elevated in individuals with prodromal dementia, while none except Abeta were elevated in aged APPswe mice. These results are consistent with the amyloid hypothesis of AD and extend it to include widespread protein insolubility, not exclusively Abeta insolubility, early in AD pathogenesis even before the onset of clinical dementia.

Enhancement of scutellarin oral delivery efficacy by vitamin B12-modified amphiphilic chitosan derivatives to treat type II diabetes induced-retinopathy.

PubMed

Wang, Jingnan; Tan, Jiayun; Luo, Jiahao; Huang, Peilin; Zhou, Wuyi; Chen, Luming; Long, Lingli; Zhang, Li-Ming; Zhu, Banghao; Yang, Liqun; Deng, David Y B

2017-03-01

Diabetic retinopathy is the most common complication in diabetic patients relates to high expression of VEGF and microaneurysms. Scutellarin (Scu) turned out to be effective against diabetes related vascular endothelial cell dysfunction. However, its clinical applications have been limited by its low bioavailability. In this study, we formulated and characterized a novel intestinal target nanoparticle carrier based on amphiphilic chitosan derivatives (Chit-DC-VB12) loaded with scutellarin to enhance its bioavailability and then evaluated its therapeutic effect in experimental diabetic retinopathy model. Chit-DC-VB12 nanoparticles showed low toxicity toward the human colon adenocarcinoma (Caco-2) cells and zebra fish within concentration of 250 μg/ml, owing to good biocompatibility of chitosan. The scutellarin-loaded Chit-DC-VB12 nanoparticles (Chit-DC-VB12-Scu) were then prepared by self-assembly in aqueous solution. Scanning electron microscopy and dynamic light scattering analysis indicated that the Chit-DC-VB12-Scu nanoparticles were spherical particles in the sizes ranging from 150 to 250 nm. The Chit-DC-VB12-Scu nanoparticles exhibited high permeation in Caco-2 cell, indicated it could be beneficial to be absorbed in humans. We also found that Chit-DC-VB12 nanoparticles had a high cellular uptake. Bioavailability studies were performed in Sprague-Dawley rats, which present the area under the curve of scutellarin of Chit-DC-VB12-Scu was two to threefolds greater than that of free scutellarin alone. Further to assess the therapeutic efficacy of diabetic retinopathy, we showed Chit-DC-VB12-Scu down-regulated central retinal artery resistivity index and the expression of angiogenesis proteins (VEGF, VEGFR2, and vWF) of retinas in type II diabetic rats. Chit-DC-VB12 nanoparticles loaded with scutellarin have better bioavailability and cellular uptake efficiency than Scu, while Chit-DC-VB12-Scu nanoparticles alleviated the structural disorder of intraretinal neovessels in the retina induced by diabetes, and it also inhibited the retinal neovascularization via down-regulated the expression of angiogenesis proteins. In conclusion, the Chit-DC-VB12 nanoparticles enhanced scutellarin oral delivery efficacy and exhibited potential as small intestinal target promising nano-carriers for treatment of type II diabetes induced-retinopathy.

Microspectroscopic analysis of green fluorescent proteins infiltrated into mesoporous silica nanochannels.

PubMed

Ma, Yujie; Rajendran, Prayanka; Blum, Christian; Cesa, Yanina; Gartmann, Nando; Brühwiler, Dominik; Subramaniam, Vinod

2011-04-01

The infiltration of enhanced green fluorescent protein (EGFP) into nanochannels of different diameters in mesoporous silica particles was studied in detail by fluorescence microspectroscopy at room temperature. Silica particles from the MCM-41, ASNCs and SBA-15 families possessing nanometer-sized (3-8 nm in diameter) channels, comparable to the dimensions of the infiltrated guest protein EGFP (barrel structure with dimensions of 2.4 nm × 4.2 nm), were used as hosts. We found that it is necessary to first functionalize the surfaces of the silica particles with an amino-silane for effective encapsulation of EGFP. We demonstrated successful infiltration of the protein into the nanochannels based on fluorescence microspectroscopy and loading capacity calculations, even for nanochannel diameters approaching the protein dimensions. We studied the spatial distributions of the EGFPs within the silica particles by confocal laser scanning microscopy (CLSM) and multimode microscopy. Upon infiltration, the fluorescence lifetime drops as expected for an emitter embedded in a high refractive index medium. Further, the spectral properties of EGFP are preserved, confirming the structural integrity of the infiltrated protein. This inorganic-protein host-guest system is an example of a nanobiophotonic hybrid system that may lead to composite materials with novel optical properties. Copyright © 2010 Elsevier Inc. All rights reserved.

Kinetics of temperature response of PEO-b-PNIPAM-b-PAA triblock terpolymer aggregates and of their complexes with lysozyme

DOE PAGES

Papagiannopoulos, Aristeidis; Meristoudi, Anastasia; Hong, Kunlun; ...

2015-12-18

We present the kinetics of temperature response of a PEO-b-PNIPAM-b-PAA triblock terpolymer and of its complexes with lysozyme in aqueous solution. It is found that during the coil-to-globule transition of PNIPAM new bonds within the polymer aggregates are created, making the transition of the aggregates partially irreversible. This effect is also found for the protein loaded PEO-b-PNIPAM-b-PAA aggregates whereas in this case protein globules appear to enhance the formation of bonds, making the transition totally irreversible. The internal dynamics of both aggregates and complexes are “frozen” once the temperature is increased upon PINIPAM's LCST in water and remain so evenmore » when the temperature drops below LCST. As a result, we investigate the complexation kinetics of lysozyme and PEO-b-PNIPAM-b-PAA and observe that it occurs in two stages, one where protein globules adsorb on single pre-formed aggregates and one where protein globules cause inter-aggregate clustering.« less

Kinetics of temperature response of PEO-b-PNIPAM-b-PAA triblock terpolymer aggregates and of their complexes with lysozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papagiannopoulos, Aristeidis; Meristoudi, Anastasia; Hong, Kunlun

We present the kinetics of temperature response of a PEO-b-PNIPAM-b-PAA triblock terpolymer and of its complexes with lysozyme in aqueous solution. It is found that during the coil-to-globule transition of PNIPAM new bonds within the polymer aggregates are created, making the transition of the aggregates partially irreversible. This effect is also found for the protein loaded PEO-b-PNIPAM-b-PAA aggregates whereas in this case protein globules appear to enhance the formation of bonds, making the transition totally irreversible. The internal dynamics of both aggregates and complexes are “frozen” once the temperature is increased upon PINIPAM's LCST in water and remain so evenmore » when the temperature drops below LCST. As a result, we investigate the complexation kinetics of lysozyme and PEO-b-PNIPAM-b-PAA and observe that it occurs in two stages, one where protein globules adsorb on single pre-formed aggregates and one where protein globules cause inter-aggregate clustering.« less

Mechanism-Based Enhanced Delivery of Drug-Loaded Targeted Nanoparticles for Breast Cancer Therapy

DTIC Science & Technology

2014-02-01

Enhanced Delivery of Drug-Loaded Targeted Nanoparticles for Breast Cancer Therapy” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-11-1-0166 5c... Nanotechnologies in Living Systems”, Moscow Region, Russia, September, 2011. 3. “Ionic nanogels for drug delivery in cancer ”. NanoDDS’12; Atlantic City, New...AD Award Number: W81XWH-11-1-0166 TITLE: Mechanism-Based Enhanced Delivery of Drug-Loaded Targeted Nanoparticles for Breast

Mechanism-Based Enhanced Delivery of Drug-Loaded Targeted Nanoparticles for Breast Cancer Therapy

DTIC Science & Technology

2014-02-01

Based Enhanced Delivery of Drug-Loaded Targeted Nanoparticles for Breast Cancer Therapy” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-11-1-0167 5c... Nanotechnologies in Living Systems”, Moscow Region, Russia, September, 2011. 3. “Ionic nanogels for drug delivery in cancer ”. NanoDDS’12; Atlantic City, New...AD Award Number: W81XWH-11-1-0167 TITLE: Mechanism-Based Enhanced Delivery of Drug-Loaded Targeted Nanoparticles for Breast

Cell type-selective disease-association of genes under high regulatory load.

PubMed

Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

2015-10-15

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bioinspired Diselenide-Bridged Mesoporous Silica Nanoparticles for Dual-Responsive Protein Delivery.

PubMed

Shao, Dan; Li, Mingqiang; Wang, Zheng; Zheng, Xiao; Lao, Yeh-Hsing; Chang, Zhimin; Zhang, Fan; Lu, Mengmeng; Yue, Juan; Hu, Hanze; Yan, Huize; Chen, Li; Dong, Wen-Fei; Leong, Kam W

2018-05-28

Controlled delivery of protein therapeutics remains a challenge. Here, the inclusion of diselenide-bond-containing organosilica moieties into the framework of silica to fabricate biodegradable mesoporous silica nanoparticles (MSNs) with oxidative and redox dual-responsiveness is reported. These diselenide-bridged MSNs can encapsulate cytotoxic RNase A into the 8-10 nm internal pores via electrostatic interaction and release the payload via a matrix-degradation controlled mechanism upon exposure to oxidative or redox conditions. After surface cloaking with cancer-cell-derived membrane fragments, these bioinspired RNase A-loaded MSNs exhibit homologous targeting and immune-invasion characteristics inherited from the source cancer cells. The efficient in vitro and in vivo anti-cancer performance, which includes increased blood circulation time and enhanced tumor accumulation along with low toxicity, suggests that these cell-membrane-coated, dual-responsive degradable MSNs represent a promising platform for the delivery of bio-macromolecules such as protein and nucleic acid therapeutics. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A simple theory of motor protein kinetics and energetics. II.

PubMed

Qian, H

2000-01-10

A three-state stochastic model of motor protein [Qian, Biophys. Chem. 67 (1997) pp. 263-267] is further developed to illustrate the relationship between the external load on an individual motor protein in aqueous solution with various ATP concentrations and its steady-state velocity. A wide variety of dynamic motor behavior are obtained from this simple model. For the particular case of free-load translocation being the most unfavorable step within the hydrolysis cycle, the load-velocity curve is quasi-linear, V/Vmax = (cF/Fmax-c)/(1-c), in contrast to the hyperbolic relationship proposed by A.V. Hill for macroscopic muscle. Significant deviation from the linearity is expected when the velocity is less than 10% of its maximal (free-load) value--a situation under which the processivity of motor diminishes and experimental observations are less certain. We then investigate the dependence of load-velocity curve on ATP (ADP) concentration. It is shown that the free load Vmax exhibits a Michaelis-Menten like behavior, and the isometric Fmax increases linearly with ln([ATP]/[ADP]). However, the quasi-linear region is independent of the ATP concentration, yielding an apparently ATP-independent maximal force below the true isometric force. Finally, the heat production as a function of ATP concentration and external load are calculated. In simple terms and solved with elementary algebra, the present model provides an integrated picture of biochemical kinetics and mechanical energetics of motor proteins.

Folic acid-conjugated soybean protein-based nanoparticles mediate efficient antitumor ability in vitro.

PubMed

Yao, Weijing; Zha, Qian; Cheng, Xu; Wang, Xin; Wang, Jun; Tang, Rupei

2016-11-23

In this study, soy protein isolate was hydrolyzed by compound enzymes to give aqueous soy protein with low molecular weights. Folic acid modified and free soy protein nanoparticles were successfully prepared by a desolvation method as target-specific drug delivery, respectively. Ultraviolet spectrophotometry demonstrated that folic acid was successfully grafted onto soy protein. The shape and size of folic acid modified soy protein nanoparticles were detected by transmission electron microscopy, scanning electron microscope, and dynamic light scattering. In addition, a series of characteristics including kinetic stability, pH stability, and time stability were also performed. Doxorubicin was successfully loaded into folic acid modified soy protein nanoparticles, and the encapsulation and loading efficiencies were 96.7% and 23%, respectively. Doxorubicin-loaded folic acid modified soy protein nanoparticles exhibited faster drug release rate than soy protein nanoparticles in PBS solution (pH = 5). The tumor penetration and antitumor experiments were done using three-dimensional multicellular tumor spheroids as the in vitro model. The results proved that folic acid modified soy protein nanoparticles display higher penetration and accumulation than soy protein nanoparticles, therefore possessing efficient growth inhibitory ability against multicellular tumor spheroids. © The Author(s) 2016.

Lightening the load: perceptual load impairs visual detection in typical adults but not in autism.

PubMed

Remington, Anna M; Swettenham, John G; Lavie, Nilli

2012-05-01

Autism spectrum disorder (ASD) research portrays a mixed picture of attentional abilities with demonstrations of enhancements (e.g., superior visual search) and deficits (e.g., higher distractibility). Here we test a potential resolution derived from the Load Theory of Attention (e.g., Lavie, 2005). In Load Theory, distractor processing depends on the perceptual load of the task and as such can only be eliminated under high load that engages full capacity. We hypothesize that ASD involves enhanced perceptual capacity, leading to the superior performance and increased distractor processing previously reported. Using a signal-detection paradigm, we test this directly and demonstrate that, under higher levels of load, perceptual sensitivity was reduced in typical adults but not in adults with ASD. These findings confirm our hypothesis and offer a promising solution to the previous discrepancies by suggesting that increased distractor processing in ASD results not from a filtering deficit but from enhanced perceptual capacity.

Confocal Raman characterization of different protein desorption behaviors from chromatographic particles.

PubMed

Xiao, Yuewu; Stone, Thomas; Moya, Wilson; Killian, Paul; Herget, Thomas

2014-01-21

Confocal Raman spectroscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure protein desorption from chromatographic particles. Monoclonal antibody was loaded onto the Fractogel EMD SO3 (M) cation exchanger at either pH 5 or pH 4. Confocal Raman measurement suggests that only the protein loaded at pH 5 is able to release from chromatographic particles in the elution buffer. Detailed comparison of high-quality spectra indicates that, while proteins loaded at both pH values showed a predominant β-sheet conformation, protein loaded at pH 4 has a broader amide I band with more intensity in the >1680 cm(-1) region. This small but clear and reproducible amide I bandwidth increase is not observed for protein in the solution state at pH 4. No definitive assignment of the increased Raman intensity in the >1680 cm(-1) region could be made, but it might be related to structural changes involved in the association of protein molecules in the adsorbed state, which helps to explain the nearly 100% retention under elution conditions of the monoclonal antibody adsorbed at pH 4 in chromatographic particles.

Correlating bioaerosol load with PM2.5 and PM10cf concentrations: a comparison between natural desert and urban-fringe aerosols

NASA Astrophysics Data System (ADS)

Boreson, Justin; Dillner, Ann M.; Peccia, Jordan

2004-11-01

Seasonal allergies and microbial mediated respiratory diseases, can coincide with elevated particulate matter concentrations, often when dry desert soils are disturbed. In addition to effects from the allergens, allergic and asthmatic responses may be enhanced when chemical and biological constituents of particulate matter (PM) are combined together. Because of these associations and also the recent regulatory and health-related interests of monitoring PM2.5, separately from total PM10, the biological loading between the fine (dp<2.5 μm) and coarse (2.5 μm

Lactoferrin modulation of BCG-infected dendritic cell functions

PubMed Central

Hwang, Shen-An

2009-01-01

Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-2] and increased regulatory cytokine, transforming growth factor-β1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN-γ activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3+CD4+ splenocytes, as defined by increasing IFN-γ production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN-γ producing CD3+CD4+CD62L− splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells. PMID:19692539

Immunogenicity and efficacy of immunodeficiency virus-like particles pseudotyped with the G protein of vesicular stomatitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuate, Seraphin; Stahl-Hennig, Christiane; Stoiber, Heribert

2006-07-20

Vaccination with exogenous antigens such as recombinant viral proteins, immunodeficiency virus-derived whole inactivated virus particles, or virus-like particles (VLP) has generally failed to provide sufficient protection in animal models for AIDS. Pseudotyping VLPs with the vesicular stomatitis virus G protein (VSV-G), which is known to mediate entry into dendritic cells, might allow more efficient stimulation of immune responses. Therefore, we pseudotyped noninfectious immunodeficiency virus-like particles with VSV-G and carried out a preliminary screen of their immunogenicity and vaccination efficacy. Incorporation of VSV-G into HIV-1 VLPs led to hundred-fold higher antibody titers to HIV-1 Gag and enhancement of T cell responsesmore » in mice. Repeated vaccination of rhesus monkeys for 65 weeks with VSV-G pseudotyped simian immunodeficiency virus (SIV)-like particles (VLP[G]) provided initial evidence for efficient suppression of viral load after mucosal challenge with the SIVmac239 virus. Challenge of monkeys after a 28 week vaccination regimen with VLP[G] led to a reduction in peak viremia, but persistent suppression of viral load was not achieved. Due to limitations in the number of animals available for this study, improved efficacy of VSV-G pseudotyped VLPs in nonhuman primates could not be demonstrated. However, mouse experiments revealed that pseudotyping of VLPs with fusion-competent VSV-G clearly improves their immunogenicity. Additional strategies, particularly adjuvants, should be considered to provide greater protection against a challenge with pathogenic immunodeficiency virus.« less

Exosomes: Tunable Nano Vehicles for Macromolecular Delivery of Transferrin and Lactoferrin to Specific Intracellular Compartment.

PubMed

Malhotra, Himanshu; Sheokand, Navdeep; Kumar, Santosh; Chauhan, Anoop S; Kumar, Manoj; Jakhar, Priyanka; Boradia, Vishant M; Raje, Chaaya I; Raje, Manoj

2016-05-01

Due to their abundant ubiquitous presence, rapid uptake and increased requirement in neoplastic tissue, the delivery of the iron carrier macromolecules transferrin (Tf) and lactoferrin (Lf) into mammalian cells is the subject of intense interest for delivery of drugs and other target molecules into cells. Utilizing exosomes obtained from cells of diverse origin we confirmed the presence of the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has recently been characterized as a Tf and Lf receptor. Using a combination of biochemical, biophysical and imaging based methodologies, we demonstrate that GAPDH present in exosomes captures Tf and Lf and subsequently effectively delivers these proteins into mammalian cells. Exosome vesicles prepared had a size of 51.2 ± 23.7 nm. They were found to be stable in suspension with a zeta potential (ζ-potential) of -28.16 ± 1.15 mV. Loading of Tf/Lf did not significantly affect ζ-potential of the exosomes. The carrier protein loaded exosomes were able to enhance the delivery of Tf/Lf by 2 to 3 fold in a diverse panel of cell types. Ninety percent of the internalized cargo via this route was found to be specifically delivered into late endosome and lysosomes. We also found exosomes to be tunable nano vehicles for cargo delivery by varying the amount of GAPDH associated with exosome. The current study opens a new avenue of research for efficient delivery of these vital iron carriers into cells employing exosomes as a nano delivery vehicle.

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies.

PubMed

Kumar, Rajeev; Wyman, Charles E

2009-09-01

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and beta-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase-xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

SANS contrast variation study of magnetoferritin structure at various iron loading

NASA Astrophysics Data System (ADS)

Melnikova, Lucia; Petrenko, Viktor I.; Avdeev, Mikhail V.; Ivankov, Oleksandr I.; Bulavin, Leonid A.; Garamus, Vasil M.; Almásy, László; Mitroova, Zuzana; Kopcansky, Peter

2015-03-01

Magnetoferritin, a synthetic derivate of iron storage protein - ferritin, has been synthesized with different iron oxide loading values. Small-angle neutron scattering experiments were applied to study the structure of magnetoferritin solutions using contrast variation method by varying the light to heavy water ratio of the solvent. Higher iron loading leads to increase of the neutron scattering length density of magnetoferritin and also to the increase of the polydispersity of complexes. The formation of the magnetic core and the variation of the protein shell structure upon iron loading are concluded.

Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

NASA Technical Reports Server (NTRS)

Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

1980-01-01

Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

A Proteomics Approach to the Protein Normalization Problem: Selection of Unvarying Proteins for MS-Based Proteomics and Western Blotting.

PubMed

Wiśniewski, Jacek R; Mann, Matthias

2016-07-01

Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.

Cancer-targeting siRNA delivery from porous silicon nanoparticles.

PubMed

Wan, Yuan; Apostolou, Sinoula; Dronov, Roman; Kuss, Bryone; Voelcker, Nicolas H

2014-10-01

Porous silicon nanoparticles (pSiNPs) with tunable pore size are biocompatible and biodegradable, suggesting that they are suitable biomaterials as vehicles for drug delivery. Loading of small interfering RNA (siRNA) into the pores of pSiNPs can protect siRNA from degradation as well as improve the cellular uptake. We aimed to deliver MRP1 siRNA loaded into pSiNPs to glioblastoma cells, and to demonstrate downregulation of MRP1 at the mRNA and protein levels. 50-220 nm pSiNPs with an average pore size of 26 nm were prepared, followed by electrostatic adsorption of siRNA into pores. Oligonucleotide loading and release profiles were investigated; MRP1 mRNA and protein expression, cell viability and cell apoptosis were studied. Approximately 7.7 µg of siRNA was loaded per mg of pSiNPs. Cells readily took up nanoparticles after 30 min incubation. siRNA-loaded pSiNPs were able to effectively downregulate target mRNA (~40%) and protein expression (31%), and induced cell apoptosis and necrosis (33%). siRNA loaded pSiNPs downregulated mRNA and protein expression and induced cell death. This novel siRNA delivery system may pave the way towards developing more effective tumor therapies.

Model studies of diffusion-controlled (2-hydroxyethyl methacrylate) HEMA hydrogel membranes for controlled release of proteins

NASA Astrophysics Data System (ADS)

Appawu, Jennifer A. M.

This thesis project consisted of three main components that were connected by roots in chemical analysis for studies in tissue engineering. The first part focused on characterizing the structural parameters of synthetic cross-linked poly (2-hydroxyethyl methacrylate) (Poly(HEMA) hydrogel membranes to determine optimal formulations for clinical studies. Poly(HEMA) membranes were loaded with Keratincocyte Growth Factor (KGF) for controlled release studies. Protein loading and release kinetics were determined with fluorescence spectroscopy. The spatial distribution of a protein in the membrane was determined using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). The last part of the project focused on determining the biological effects of the polymer membranes in-vitro with a model cell line and a pilot in-vivo animal study. Based on the components completed in this project, five chapters are included in this dissertation document and are summarized below. A new protocol was developed using fluorescence spectroscopy that measured the rate of protein diffusion into cross-linked polymer membranes by measuring the change in the fluorescence intensity of the protein solution. This technique was also able to detect a conformational change that occurs within protein when KGF was imbibed within these cross-linked polymer membranes. ToF-SIMS chemical imaging and 3D depth profiling was used to determine the spatial distribution of KGF protein in frozen-hydrated HEMA hydrogel membranes. The 3D depth profiles showed that the KGF protein was aggregated in bright spots that indicated that KGF was not spatially homogenous on the surface and through the depth profiles. 3D depth profiles of the membranes studied at various times during release studies show that areas with aggregated proteins were retained during release, and at times with maximum release. The interpretation of the bright regions is that the KGf protein interacted with the cross-linked network of the hydrogel membranes, making it not available for release. The in-vitro biological experiments with the HaCaT cell line showed that the HEMA hydrogels were capable of sustaining cell viability, proliferation, and adhesion through cell adhesion and wounding experiments. The pilot in-vivo animal study also revealed that KGF protein had retained its pharmacological activity. The study also showed that the KGF protein enhanced the rate of wound closure.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue.

PubMed

Pennington, Kyla; McGregor, Emma; Beasley, Clare L; Everall, Ian; Cotter, David; Dunn, Michael J

2004-01-01

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.

Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes.

PubMed

Li, Xiang; Yang, Weijing; Zou, Yan; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan

2015-12-28

Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157-175 nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1-24.5 wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6 nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid deposition of triangular silver nanoplates on planar surfaces: application to metal-enhanced fluorescence.

PubMed

Aslan, Kadir; Lakowicz, Joseph R; Geddes, Chris D

2005-04-07

A simple and rapid wet-chemical technique for the deposition of silver triangles on conventional glass substrates, which alleviates the need for lithography, has been developed. The technique is based on the seed-mediated cetyltrimethylammonium-bromide-directed growth of silver triangles on glass surfaces, where smaller spherical silver seeds that were attached to the surface were subsequently converted and grown into silver triangles in the presence of a cationic surfactant and silver ions. The size of the silver triangles was controlled by sequential immersion of silver seed-coated glass substrates into a growth solution and by the duration time of immersion. Atomic force microscopy studies revealed that the size of the silver triangles ranged between 100 and 500 nm. Interestingly, these new surfaces are a significant improvement over traditional silver island films for applications in metal-enhanced fluorescence. A routine 16-fold enhancement in emission intensity was typically observed, for protein-immobilized indocyanine green, with a relatively very low loading density of silver triangles on the glass surface.

Dietary protein and skeletal health: a review of recent human research.

PubMed

Kerstetter, Jane E; Kenny, Anne M; Insogna, Karl L

2011-02-01

Both dietary calcium and vitamin D are undoubtedly beneficial to skeletal health. In contrast, despite intense investigation, the impact of dietary protein on calcium metabolism and bone balance remains controversial. A widely held view is that high intakes of animal protein result in increased bone resorption, reduced bone mineral density, and increased fractures because of its ability to generate a high fixed metabolic acid load. The purpose of this review is to present the recent or most important epidemiological and clinical trials in humans that evaluated dietary protein's impact on skeletal health. Many epidemiological studies have found a significant positive relationship between protein intake and bone mass or density. Similarly, isotopic studies in humans have also demonstrated greater calcium retention and absorption by individuals consuming high-protein diets, particularly when the calcium content of the diet was limiting. High-protein intake may positively impact bone health by several mechanisms, including calcium absorption, stimulation of the secretion of insulin-like growth factor-1, and enhancement of lean body mass. The concept that an increase in dietary protein induces a large enough shift in systemic pH to increase osteoclastic bone resorption seems untenable. Recent epidemiological, isotopic and meta-analysis studies suggest that dietary protein works synergistically with calcium to improve calcium retention and bone metabolism. The recommendation to intentionally restrict dietary protein to improve bone health is unwarranted, and potentially even dangerous to those individuals who consume inadequate protein.

Load controller and method to enhance effective capacity of a photovotaic power supply using a dynamically determined expected peak loading

DOEpatents

Perez, Richard

2003-04-01

A load controller and method are provided for maximizing effective capacity of a non-controllable, renewable power supply coupled to a variable electrical load also coupled to a conventional power grid. Effective capacity is enhanced by monitoring power output of the renewable supply and loading, and comparing the loading against the power output and a load adjustment threshold determined from an expected peak loading. A value for a load adjustment parameter is calculated by subtracting the renewable supply output and the load adjustment parameter from the current load. This value is then employed to control the variable load in an amount proportional to the value of the load control parameter when the parameter is within a predefined range. By so controlling the load, the effective capacity of the non-controllable, renewable power supply is increased without any attempt at operational feedback control of the renewable supply. The expected peak loading of the variable load can be dynamically determined within a defined time interval with reference to variations in the variable load.

Micro-emulsification/encapsulation of krill oil by complex coacervation with krill protein isolated using isoelectric solubilization/precipitation.

PubMed

Shi, Liu; Beamer, Sarah K; Yang, Hong; Jaczynski, Jacek

2018-04-01

This study determined feasibility of krill protein isolated with isoelectric solubilization/precipitation (ISP) as wall material to microencapsulate krill oil by freeze-drying. Effects of krill oil/krill protein ratio on properties of microcapsules were investigated. With increased ratio, crude protein of microcapsules decreased, while total lipid increased. Although microcapsule oil loading capacity increased, loading and encapsulation efficiencies decreased. Thin layer chromatography (TLC) confirmed abundance of phospholipids, which are amphiphilic; and thus, resulted in stable emulsion (emulsion stability index). Microcapsules contained ω-3 polyunsaturated fatty acids (PUFAs) at 43-60, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at 28-41 and 9-11 g/100g of total FAs, respectively. SDS-PAGE electrophoresis revealed proteolysis of ISP krill protein, probably causing reduced loading and encapsulation efficiencies. SEM showed that krill oil/krill protein ratio affected surface microstructure. ISP krill protein showed potential as a wall material to microencapsulate krill oil; and thus, expand application of krill oil/protein for human consumption. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

NASA Technical Reports Server (NTRS)

Hammond, Dianne K.; Becker, Jeanne; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

NASA Technical Reports Server (NTRS)

Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Identification of ovarian cancer-associated proteins in symptomatic women: A novel method for semi-quantitative plasma proteomics.

PubMed

Shield-Artin, Kristy L; Bailey, Mark J; Oliva, Karen; Liovic, Ana K; Barker, Gillian; Dellios, Nicole L; Reisman, Simone; Ayhan, Mustafa; Rice, Gregory E

2012-04-01

To evaluate the utility of an enhanced biomarker discovery approach in order to identify potential biomarkers relevant to ovarian cancer detection. We combined immuno-depletion, liquid-phase IEF, 1D-DIGE, MALDI-TOF/MS and LC-MS/MS to identify differentially expressed proteins in the plasma of symptomatic ovarian cancer patients, stratified by stage, compared to samples obtained from normal subjects. We demonstrate that this approach is a practical alternative to traditional 2D gel techniques and that it has some advantages, most notably increased protein capacity. Proteins were identified in all 76 bands excised from the gels in this project and confirmed the cancer-associated expression of several well-established biomarkers of ovarian cancer. These included C-reactive protein (CRP), haptoglobin, alpha-2 macroglobulin and A1A2. We also identified new ovarian cancer candidate biomarkers, Protein S100-A9 (S100A9) and multimerin-2. The cancer-associated differential expression of CRP and S100A9 was further confirmed by Western blot and ELISA. The methods developed in this study allow for the increased loading of plasma proteins into the analytical stream when compared to traditional 2D-DIGE. This increased protein identification sensitivity allowed us to identify new putative ovarian cancer biomarkers. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Load controller and method to enhance effective capacity of a photovoltaic power supply

DOEpatents

Perez, Richard

2000-01-01

A load controller and method are provided for maximizing effective capacity of a non-controllable, renewable power supply coupled to a variable electrical load also coupled to a conventional power grid. Effective capacity is enhanced by monitoring power output of the renewable supply and loading, and comparing the loading against the power output and a load adjustment threshold determined from an expected peak loading. A value for a load adjustment parameter is calculated by subtracting the renewable supply output and the load adjustment parameter from the current load. This value is then employed to control the variable load in an amount proportional to the value of the load control parameter when the parameter is within a predefined range. By so controlling the load, the effective capacity of the non-controllable, renewable power supply is increased without any attempt at operational feedback control of the renewable supply. The renewable supply may comprise, for example, a photovoltaic power supply or a wind-based power supply.

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia.

PubMed

Packiriswamy, Nandakumar; Steury, Michael; McCabe, Ian C; Fitzgerald, Scott D; Parameswaran, Narayanan

2016-05-01

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

PubMed Central

Ran, Yong; Fanucci, Gail E.

2009-01-01

Abstract The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles. PMID:19580763

Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.

PubMed

Ran, Yong; Fanucci, Gail E

2009-07-08

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.

Nitrogen-Doped Carbon Nanotubes Supported by Macroporous Carbon as an Efficient Enzymatic Biosensing Platform for Glucose.

PubMed

Song, Yonghai; Lu, Xingping; Li, Yi; Guo, Qiaohui; Chen, Shuiliang; Mao, Lanqun; Hou, Haoqing; Wang, Li

2016-01-19

Effective immobilization of enzymes/proteins on an electrode surface is very essential for biosensor development, but it still remains challenging because enzymes/proteins tend to form close-packed structures on the electrode surface. In this work, nitrogen-doped carbon nanotubes (NCNTs) supported by three-dimensional Kenaf Stem-derived porous carbon (3D-KSC) (denoted as 3D-KSC/NCNTs) nanocomposites were constructed as the supporting matrix to load glucose oxidase (GOD) for preparing integrated glucose biosensors. These NCNTs are vertically arrayed on the channel walls of the 3D-KSC via the chemical vapor deposition method, which could noticeably increase the effective surface area, mechanical stability, and active sites (originating from the doped nitrogen) of the nanocomposites. The integrated glucose biosensor exhibits some advantages over the traditional GOD electrodes in terms of the capability to promote the direct electron transfer of GOD, enhance the mechanical stability of the biosensor attributed to the strong interaction between NCNTs and GOD, and enlarge the specific surface area to efficiently load a large number of GODs. The as-prepared biosensor shows a good performance toward both oxygen reduction and glucose biosensing. This study essentially offers a novel approach for the development of biosensors with excellent analytical properties.

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer’s disease

PubMed Central

Alfonso, Stephanie I.; Callender, Julia A.; Hooli, Basavaraj; Antal, Corina E.; Mullin, Kristina; Sherman, Mathew A.; Lesné, Sylvain E.; Leitges, Michael; Newton, Alexandra C.; Tanzi, Rudolph E.; Malinow, Roberto

2016-01-01

Alzheimer’s disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-β (Aβ) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA−/− neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme. PMID:27165780

Clostridium difficile the hospital plague.

PubMed

Czepiel, J; Kozicki, M; Panasiuk, P; Birczyńska, M; Garlicki, A; Wesełucha-Birczyńska, A

2015-04-07

Clostridium difficile infection (CDI) has become one of the major public health threats in the last two decades. An increase has been observed not only in the rate of CDI, but also in its severity and mortality. Symptoms caused by this pathogen are accompanied by intense local and systemic inflammation. We confirmed that Raman microspectroscopy can help us in understanding CDI pathogenesis. A single erythrocyte of patients with CDI shows a difference, approximately 10 times, in the intensity of the Raman spectra at the beginning of hospitalization and after one week of treatment. The intensity level is an indicator of the spread of the inflammation within the cell, confirmed by standard laboratory tests. Many of the observed bands with enormously enhanced intensity, e.g. 1587, 1344, 1253, 1118 and 664 cm(-1), come from the symmetric vibration of the pyrrole ring. Heme variation of recovered cells in the acute CDI state between the first and the seventh day of treatment seems to show increased levels of oxygenated hemoglobin. Intense inflammation alters the conformation of the protein which is reflected in the significant changes in the amide I, II and III bands. There is an observed shift and a significant intensity increase of 1253 and 970 cm(-1) amide III and skeletal protein backbone CC stretching vibration bands, respectively. Principal Component Analysis (PCA) was used to find the variance in the data collected on the first and seventh day. PC2 loading in the 1645-1500 cm(-1) range shows an increase of heme, Tyr, Trp, or Phe vibrations because of changes in the protein microenvironment due to their exposure. Positive maxima at 1621, 1563 and 1550 in the PC2 loading originated from the ring vibrations. These observations indicate that Clostridium difficile toxins induce cytopathogenicity by altering cellular proteins.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

PubMed

Lalli, M J; Yong, J; Prasad, V; Hashimoto, K; Plank, D; Babu, G J; Kirkpatrick, D; Walsh, R A; Sussman, M; Yatani, A; Marbán, E; Periasamy, M

2001-07-20

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

PubMed

Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

2017-07-01

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic core-shell heparin-PEG microparticles presented here overcome this limitation by sequestering proteins through a PEG-based shell onto a protein-protective heparin core, temporarily isolating bound proteins from the cellular microenvironment, and re-delivering proteins only after degradation of the PEG-based shell. Thus, these core-shell microparticles have potential to be a novel tool to harness and isolate proteins produced in the cellular environment and then control when proteins are re-introduced for the most effective tissue regeneration and repair. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Photothermal enhancement of chemotherapy mediated by gold-silica nanoshell-loaded macrophages: in vitro squamous cell carcinoma study

NASA Astrophysics Data System (ADS)

Madsen, Steen J.; Shih, En-Chung; Peng, Qian; Christie, Catherine; Krasieva, Tatiana; Hirschberg, Henry

2016-01-01

Moderate hyperthermia (MHT) has been shown to enhance the effects of chemotherapeutic agents in a wide variety of cancers. The purpose of this study was to investigate the combined effects of commonly used chemotherapeutic agents with MHT induced by near-infrared (NIR) activation of gold nanoshell (AuNS)-loaded macrophages (Ma). AuNS-loaded murine Ma combined with human FaDu squamous cells, in hybrid monolayers, were subjected to three cytotoxic drugs (doxorubicin, bleomycin, cisplatin) with or without NIR laser irradiation. For all three drugs, efficacy was increased by NIR activation of AuNS-loaded Ma. The results of this in vitro study provide proof-of-concept for the use of AuNS-loaded Ma for photothermal enhancement of the effects of chemotherapy on squamous cell carcinoma.

Formulation, characteristics and antiatherogenic bioactivities of CD36-targeted epigallocatechin gallate (EGCG)-loaded nanoparticles.

PubMed

Zhang, Jia; Nie, Shufang; Martinez-Zaguilan, Raul; Sennoune, Souad R; Wang, Shu

2016-04-01

Intimal macrophages are determinant cells for atherosclerotic lesion formation by releasing inflammatory factors and taking up oxidized low-density lipoprotein (oxLDL) via scavenger receptors, primarily the CD36 receptor. (-)-Epigallocatechin-3-gallate (EGCG) has a potential to decrease cholesterol accumulation and inflammatory responses in macrophages. We made EGCG-loaded nanoparticles (Enano) using phosphatidylcholine, kolliphor HS15, alpha-tocopherol acetate and EGCG. 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), a CD36-targeted ligand found on oxLDL, was incorporated on the surface of Enano to make ligand-Enano (L-Enano). The objectives of this study are to deliver EGCG to macrophages via CD36-targeted L-Enano and to determine its antiatherogenic bioactivities. The optimized nanoparticles obtained in our study were spherical and around 108 nm in diameter, and had about 10% of EGCG loading capacity and 96% of EGCG encapsulation efficiency. Compared to Enano, CD36-targeted L-Enano had significantly higher binding affinity to and uptake by macrophages at the same pattern as oxLDL. CD36-targeted L-Enano dramatically improved EGCG stability, increased macrophage EGCG content, delivered EGCG to macrophage cytosol and avoided lysosomes. L-Enano significantly decreased macrophage mRNA levels and protein secretion of monocyte chemoattractant protein 1, but did not significantly change macrophage cholesterol content. The innovative CD36-targeted nanoparticles may facilitate targeted delivery of diagnostic, preventive and therapeutic compounds to intimal macrophages for the diagnosis, prevention and treatment of atherosclerosis with enhanced efficacy and decreased side effects. Copyright © 2015 Elsevier Inc. All rights reserved.

Consuming Lower-Protein Nutrition Bars with Added Leucine Elicits Postprandial Changes in Appetite Sensations in Healthy Women.

PubMed

Bolster, Douglas R; Rahn, Maike; Kamil, Alison G; Bristol, Lindsey T; Goltz, Shellen R; Leidy, Heather J; Blaze Mt, Melvin; Nunez, Michael A; Guo, Elizabeth; Wang, Jianquan; Harkness, Laura S

2018-04-20

Higher-protein meals (>25 g protein/meal) have been associated with enhanced satiety but the role of amino acids is unclear. Leucine has been proposed to stimulate satiety in rodents but has not been assessed in humans. We assessed the acute effects of lower-protein nutrition bars, enhanced with a leucine peptide (LP), on postprandial appetite sensations in combination with plasma leucine and peptide YY (PYY) in healthy women. Utilizing a double-blind randomized crossover design, 40 healthy women [28 ± 7.5 y; body mass index (BMI, in kg/m2): 23.5 ± 2.4] consumed the following isocaloric (180 kcal) pre-loads on 3 separate visits: control bar [9 g protein with 0 g added LP (0-g LP)] or treatment bars [11 g protein with 2 g added LP (2-g LP) or 13 g protein with 3 g added LP (3-g LP)]. Pre- and postprandial hunger, desire to eat, prospective food consumption (PFC), fullness, and plasma leucine were assessed every 30 min for 240 min. Plasma PYY was assessed hourly for 240 min (n = 24). Main effects of time (P < 0.0001) and treatment (P < 0.03) were detected for postprandial hunger, desire to eat, PFC, and fullness. Post hoc analyses revealed that the 2-g and 3-g LP bars elicited greater increases in fullness and greater decreases in PFC compared with 0-g LP (all, P < 0.05) with no differences between the 2-g and 3-g LP bars. The 2-g bar elicited greater decreases in hunger and desire to eat compared with the 0-g LP bar (both, P ≤ 0.01), whereas 3-g LP did not. Appetite incremental areas under the curves (iAUCs) and PYY outcomes were not different between bars. A treatment × time interaction was detected for plasma leucine with increases occurring in a leucine-dose-dependent manner (P < 0.0001). Despite the dose-dependent increases in plasma leucine following the consumption of lower-protein bars enhanced with LP, only the 2-g LP bar elicited consistent postprandial changes in select appetite sensations compared with the 0-g LP bar. This study was registered on clinicaltrials.gov as NCT02091570.

Nutrition Coupled with High-Load Traditional or Low-Load Blood Flow Restricted Exercise During Human Limb Suspension

NASA Technical Reports Server (NTRS)

Hackney, K. J.; Everett, M.; Ploutz-Snyder, L. L.

2011-01-01

High-load resistance exercise (HRE) and low-load blood flow restricted (BFR) exercise have demonstrated efficacy for attenuating unloading related muscle atrophy and dysfunction. In recreational exercisers, protein consumption immediately before and/or after exercise has been shown to increase the skeletal muscle anabolic response to resistance training. PURPOSE: To compare the skeletal muscle adaptations when chocolate milk intake was coupled with HRE or low-load BFR exercise [3 d/wk] during simulated lower limb weightlessness. METHODS: Eleven subjects were counterbalanced [based on age and gender] to HRE (31 +/- 14 yr, 170 +/- 13 cm, 71 +/- 18 kg, 2M/3W) or low-load BFR exercise (31 +/- 12 yr, 169 +/- 13 cm, 66 +/- 14 kg, 2M/4W) during 30 days of unilateral lower limb suspension (ULLS). Both HRE and BFR completed 3 sets of single leg press and calf raise exercise during ULLS. BFR exercise intensity was 20% of repetition maximum (1RM) with a cuff inflation pressure of 1.3 systolic blood pressure (143 4 mmHg). Cuff pressure was maintained during all 3 sets including rest intervals (90s). HRE intensity was 75% 1RM and was performed without cuff inflation. Immediately (<10 min) before and after exercise 8 fl oz of chocolate milk (150 kcal, 2.5g total fat, 22g total carbohydrate, 8g protein) was consumed to optimize acute exercise responses in favor of muscle anabolism. ULLS analog compliance was assessed from leg skin temperature recordings and plantar accelerometry. Muscle cross-sectional area (CSA) for knee extensor and plantar flexor muscle groups were determined from analysis of magnetic resonance images using ImageJ software. 1RM strength for leg press and calf raise was assessed on the Agaton exercise system. Muscular endurance during leg press and calf raise was evaluated from the maximal number of repetitions performed to volitional fatigue using 40% of pre-ULLS 1RM. RESULTS: Steps detected by plantar acceleometry declined by 98.9% during ULLS relative to an ambulatory control period. Average skin temperature of the unloaded calf declined from 27.4 C to 26.8 C (-2.1%), while there was a slight increase (+1.1%) in skin temperature in the loaded calf (27.6 C to 27.9 C). Collectively, these measures indicate strong subject compliance with the ULLS analog. Unloaded limb work performed during leg press (1514 +/- 334 vs. 576 +/- 103) and calf raise (2886 +/- 508 vs. 1233 +/- 153) exercises sessions was greater in HRE vs. BFR, respectively. Leg press training loads were 44 +/- 7 kg in HRE compared to 11 +/- 1 kg in BFR. Similarly, calf raise training loads were 81 +/- 11 kg in HRE and 16 +/- 1 kg in BFR. Pre to post-ULLS training adaptations in the unloaded leg are shown in the table. CONCLUSION: The preliminary results of this investigation suggest when HRE is optimized for muscle anabolism during unloading muscle size and strength are preserved (or enhanced) at the expense of muscle endurance. In contrast, when BFR exercise is optimized for muscle anabolism during unloading muscle endurance is preserved (or enhanced) at the expense of muscle size and strength

Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

PubMed

Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

2017-11-01

The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Power system security enhancement through direct non-disruptive load control

NASA Astrophysics Data System (ADS)

Ramanathan, Badri Narayanan

The transition to a competitive market structure raises significant concerns regarding reliability of the power grid. A need to build tools for security assessment that produce operating limit boundaries for both static and dynamic contingencies is recognized. Besides, an increase in overall uncertainty in operating conditions makes corrective actions at times ineffective leaving the system vulnerable to instability. The tools that are in place for stability enhancement are mostly corrective and suffer from lack of robustness to operating condition changes. They often pose serious coordination challenges. With deregulation, there have also been ownership and responsibility issues associated with stability controls. However, the changing utility business model and the developments in enabling technologies such as two-way communication, metering, and control open up several new possibilities for power system security enhancement. This research proposes preventive modulation of selected loads through direct control for power system security enhancement. Two main contributions of this research are the following: development of an analysis framework and two conceptually different analysis approaches for load modulation to enhance oscillatory stability, and the development and study of algorithms for real-time modulation of thermostatic loads. The underlying analysis framework is based on the Structured Singular Value (SSV or mu) theory. Based on the above framework, two fundamentally different approaches towards analysis of the amount of load modulation for desired stability performance have been developed. Both the approaches have been tested on two different test systems: CIGRE Nordic test system and an equivalent of the Western Electric Coordinating Council test system. This research also develops algorithms for real-time modulation of thermostatic loads that use the results of the analysis. In line with some recent load management programs executed by utilities, two different algorithms based on dynamic programming are proposed for air-conditioner loads, while a decision-tree based algorithm is proposed for water-heater loads. An optimization framework has been developed employing the above algorithms. Monte Carlo simulations have been performed using this framework with the objective of studying the impact of different parameters and constraints on the effectiveness as well as the effect of control. The conclusions drawn from this research strongly advocate direct load control for stability enhancement from the perspectives of robustness and coordination, as well as economic viability and the developments towards availability of the institutional framework for load participation in providing system reliability services.

A novel synergetic targeting strategy for glioma therapy employing borneol combination with angiopep-2-modified, DOX-loaded PAMAM dendrimer.

PubMed

Han, Shunping; Zheng, Hongyue; Lu, Yanping; Sun, Yue; Huang, Anhao; Fei, Weidong; Shi, Xiaowei; Xu, Xiuling; Li, Jingjing; Li, Fanzhu

2018-01-01

Glioma is the most common primary malignant brain tumour and the effect of chemotherapy is hampered by low permeability across the blood-brain-barrier (BBB). Borneol is a time-honoured 'Guide' drug in traditional Chinese medicine and has been proved to be capable of promoting free drugs into the brain efficiently, but there are still risks that free drugs, especially anti-glioma drugs, may be disassembled and metabolised before penetrating the BBB and caused the whole brain distribution. The purpose of this paper was to investigate whether borneol intervention could facilitate the BBB penetration and assist glioma treatment by combining with doxorubicin (DOX) loaded PAMAM dendrimers drug delivery system modified with Angiopep-2 (a ligand of the low-density lipoprotein receptor-related protein, which overexpress both in the BBB and gliomas). The results demonstrated that Angiopep-2 modification could actually enhance the affinity between the dendrimers and the targeting cells and finally increase the cell uptake and boost the anti-tumour ability. Borneol physical combination could further enhance the anti-tumour efficiency of this targeting drug delivery system (TDDS) after penetrating BBB. Compared with free DOX solution, this TDDS illustrated obviously sustained and pH-dependent drug release. This suggested that this synergetic strategy provided a promising way for glioma therapy.

Elucidation of innovative antibiofilm materials.

PubMed

Marcano, Aracelys; Ba, Ousmane; Thebault, Pascal; Crétois, Raphaël; Marais, Stéphane; Duncan, Anthony C

2015-12-01

It is known for roughly a decade that bacterial communities (called biofilms) are responsible for significant enhanced antibiotherapy resistance. Biofilms are involved in tissue persistent infection, causing direct or collateral damage leading to chronic wounds development and impairing natural wound healing. In this study, we are interested in the development of supported protein materials which consist of asymmetric membranes as reservoir supports for the incorporation and controlled release of biomolecules capable of dissolving biofilms (or preventing their formation) and their use as wound dressing for chronic wound treatment. In a first step, polyhydroxyalkanoates (PHAs) asymmetric membranes were prepared using wet phase inversion technique. Scanning microscopy (SEM) analysis has showed the influence of different processing parameters. In a second step, the porous side of the membranes were functionalized with a surface treatment and then loaded with the antibiofilm agent (dispersin B). In a third step, the properties and antibiofilm performance of the loaded-membranes were evaluated. Exposure of Staphylococcus epidermidis biofilms to such systems weakly inhibited biofilm formation (weak preventive effect) but caused their detachment and disaggregation (strong curative effect). These initial results are promising since they open the way to a new generation of effective tools in the struggle against persistent bacterial infections exhibiting enhanced antibiotherapy resistance, and in particular in the case of infected chronic wounds. Copyright © 2015. Published by Elsevier B.V.

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway.

PubMed

Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

2013-11-15

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Copyright © 2013 Elsevier Inc. All rights reserved.

An Approach to the Design of a Particulate System for Oral Protein Delivery .II. Preparation and Stability Study of rhGH-Loaded Microspheres in Simulated Gastrointestinal Fluids

PubMed Central

Nafissi Varcheh, Nastaran; Aboofazeli, Reza

2011-01-01

The delivery of therapeutic proteins has gained momentum with development of biotechnology. However, large molecular weight, hydrophilic nature and susceptibility to harsh environment of gastrointestinal tract (GIT) resulted in low absorption. The main objective of this work was the design of a particulate system for oral delivery of recombinant human growth hormone (rhGH) on the basis of particle uptake mechanism in GIT. Biodegradable protein-loaded microspheres were prepared using Resomers (RG207, RG756 and RG505) by double emulsion methods. Aqueous solution of protein and freshly prepared rhGH-zinc complex were used for loading process. Various analytical methods, including fluorescence spectroscopy, SDS-PAGE electrophoresis and reversed-phase chromatography, were set up for the quantification and qualification of rhGH before and after the formulation and fabrication procedures. At the optimum conditions, microspheres were mostly below 10 μm with relatively high protein loading (> 50%). Obtained data showed that the stability of protein did not change during the formulation and microencapsulation processes. Results also showed that the encapsulation process in the presence of zinc caused no detectable change in the protein chemical stability. In-vitro stability study of microspheres in different simulated GI media indicated that the entrapped protein was physically stable. Less than 20% of rhGH was released from the microspheres incubated in both simulated stomach and intestine fluids for 3 and 6 h, respectively. PMID:24250342

PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect.

PubMed

Acharya, Sarbari; Sahoo, Sanjeeb K

2011-03-18

As mortality due to cancer continues to rise, advances in nanotechnology have significantly become an effective approach for achieving efficient drug targeting to tumour tissues by circumventing all the shortcomings of conventional chemotherapy. During the past decade, the importance of polymeric drug-delivery systems in oncology has grown exponentially. In this context, poly(lactic-co-glycolic acid) (PLGA) is a widely used polymer for fabricating 'nanoparticles' because of biocompatibility, long-standing track record in biomedical applications and well-documented utility for sustained drug release, and hence has been the centre of focus for developing drug-loaded nanoparticles for cancer therapy. Such PLGA nanoparticles have also been used to develop proteins and peptides for nanomedicine, and nanovaccines, as well as a nanoparticle-based drug- and gene-delivery system for cancer therapy, and nanoantigens and growth factors. These drug-loaded nanoparticles extravasate through the tumour vasculature, delivering their payload into the cells by the enhanced permeability and retention (EPR) effect, thereby increasing their therapeutic effect. Ongoing research about drug-loaded nanoparticles and their delivery by the EPR effect to the tumour tissues has been elucidated in this review with clarity. Copyright © 2010 Elsevier B.V. All rights reserved.

Enhanced colloidal stability, solubility and rapid dissolution of resveratrol by nanocomplexation with soy protein isolate.

PubMed

Pujara, Naisarg; Jambhrunkar, Siddharth; Wong, Kuan Yau; McGuckin, Michael; Popat, Amirali

2017-02-15

The polyphenolic compound resveratrol has received significant attention due to its many pharmacological actions such as anti-cancer, anti-inflammatory, antioxidant and antimicrobial activities. However, poor solubility and stability are major impediments for resveratrol's clinical effectiveness. In this work we have encapsulated resveratrol into soy protein isolate nanoparticles using a simple rotary evaporation technique. Resveratrol-loaded nanoparticles were around 100nm in diameter and negatively charged. Nano-encapsulated resveratrol was found to be in amorphous form and showed more than two times higher solubility with significantly increased dissolution when compared to free resveratrol. Finally, an in-vitro NF-κB inhibition assay revealed that encapsulated resveratrol was stable and retained bioactivity. This new formulation of resveratrol has the potential to boost the clinical effectiveness of this drug and could be utilised for other poorly soluble hydrophobic drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhancement and suppression in the visual field under perceptual load.

PubMed

Parks, Nathan A; Beck, Diane M; Kramer, Arthur F

2013-01-01

The perceptual load theory of attention proposes that the degree to which visual distractors are processed is a function of the attentional demands of a task-greater demands increase filtering of irrelevant distractors. The spatial configuration of such filtering is unknown. Here, we used steady-state visual evoked potentials (SSVEPs) in conjunction with time-domain event-related potentials (ERPs) to investigate the distribution of load-induced distractor suppression and task-relevant enhancement in the visual field. Electroencephalogram (EEG) was recorded while subjects performed a foveal go/no-go task that varied in perceptual load. Load-dependent distractor suppression was assessed by presenting a contrast reversing ring at one of three eccentricities (2, 6, or 11°) during performance of the go/no-go task. Rings contrast reversed at 8.3 Hz, allowing load-dependent changes in distractor processing to be tracked in the frequency-domain. ERPs were calculated to the onset of stimuli in the load task to examine load-dependent modulation of task-relevant processing. Results showed that the amplitude of the distractor SSVEP (8.3 Hz) was attenuated under high perceptual load (relative to low load) at the most proximal (2°) eccentricity but not at more eccentric locations (6 or 11°). Task-relevant ERPs revealed a significant increase in N1 amplitude under high load. These results are consistent with a center-surround configuration of load-induced enhancement and suppression in the visual field.

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

PubMed

Al-Ghobashy, Medhat A; Williams, Martin A K; Brophy, Brigid; Laible, Götz; Harding, David R K

2009-06-01

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

Analysis of pollen load based on color, physicochemical composition and botanical source.

PubMed

Modro, Anna F H; Silva, Izabel C; Luz, Cynthia F P; Message, Dejair

2009-06-01

Pollen load samples from 10 hives of Apis mellifera (L.) were analyzed based on their physicochemical composition and botanical source, considering color as a parameter for quality control. In seven samples it was possible to establish the occurrence of more than 80% of a single pollen type, characterizing them as unifloral but with protein content variation. One of the samples was exclusively composed of saprophytic fungi (Cladosporium sp.). Comparing the mean results of the fungi loads with those of the nutritional components of pollen load, the former presented higher protein, mineral matter and dry matter and lower organic matter, ethereal extract and total carbohydrate values. The monochromatic samples met the physicochemical specifications regulating pollen load quality. The results showed that homogeneous coloration of the pollen load was not found to be a good indication of unifloral pollen, confirming the importance of physicochemical analysis and melissopalynological analysis for characterization of the quality of commercial pollen load.

Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

PubMed

Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

2017-07-01

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

Development of drug delivery systems based on nanostructured porous silicon loaded with the anti-tumoral drug emodin adsorbed on silver nanoparticles

NASA Astrophysics Data System (ADS)

Hernández, Margarita; Recio, Gonzalo; Sevilla, Paz; Torres-Costa, Vicente; García-Ramos, José V.; Domingo, Concepción; Martín-Palma, Raúl J. J.

2012-10-01

A study of the fluorescence and Raman spectra of a new and complex drug delivery system formed by emodin adsorbed on silver nanoparticles embedded into a matrix of porous silicon is here reported. Several experimental methods of inclusion of the drug-silver set inside the pores, without previous functionalization of porous silicon, have been tested in order to optimize the conditions for the fluorescence detection of emodin. In this sense, we have also added bovine serum albumin to the system, finding that the presence of the protein enhances the fluores-cence signal from emodin.

Tadalafil-loaded nanostructured lipid carriers using permeation enhancers.

PubMed

Baek, Jong-Suep; Pham, Cuong Viet; Myung, Chang-Seon; Cho, Cheong-Weon

2015-11-30

Tadalafil is a phosphodiesterase-5 inhibitor indicated for the treatment of erectile dysfunction. In this study, we prepared and evaluated transdermal nanostructured lipid carriers (NLC) to improve the skin permeability of tadalafil. Tadalafil-loaded NLC dispersions were prepared using glyceryl monostearate as a solid lipid, oleic acid as a liquid lipid, and Tween 80 as a surfactant. We characterized the dispersions according to particle size, polydispersity index, zeta potential, encapsulation efficiency, and transmission electron microscopy. In vitro skin permeation studies were carried out using Franz diffusion cells, and cytotoxicity was examined using HaCaT keratinocyte cell lines. Tadalafil skin permeability increased for all tadalafil-loaded NLC formulations. The tadalafil-loaded NLC dispersion with ethanol and limonene as skin permeation enhancers exhibited the highest flux (∼4.8-fold) compared to that observed with tadalafil solution alone. Furthermore, a tadalafil-loaded NLC gel with selected permeation enhancers showed tolerance against toxicity in HaCaT cells. These results suggest that the NLC formulations with ethanol and limonene as skin permeation enhancers could be a promising dermal delivery carrier for tadalafil. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatically cross-linked injectable alginate-g-pyrrole hydrogels for neovascularization.

PubMed

Devolder, Ross; Antoniadou, Eleni; Kong, Hyunjoon

2013-11-28

Microparticles capable of releasing protein drugs are often incorporated into injectable hydrogels to minimize their displacement at an implantation site, reduce initial drug burst, and further control drug release rates over a broader range. However, there is still a need to develop methods for releasing drug molecules over extended periods of time, in order to sustain the bioactivity of drug molecules at an implantation site. In this study, we hypothesized that a hydrogel formed through the cross-linking of pyrrole units linked to a hydrophilic polymer would release protein drugs in a more sustained manner, because of an enhanced association between cross-linked pyrrole groups and the drug molecules. To examine this hypothesis, we prepared hydrogels of alginate substituted with pyrrole groups, alginate-g-pyrrole, through a horse-radish peroxidase (HRP)-activated cross-linking of the pyrrole groups. The hydrogels were encapsulated with poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with vascular endothelial growth factor (VEGF). The resulting hydrogel system released VEGF in a more sustained manner than Ca(2+) alginate or Ca(2+) alginate-g-pyrrole gel systems. Finally, implantations of the VEGF-releasing HRP-activated alginate-g-pyrrole hydrogel system on chicken chorioallantoic membranes resulted in the formation of blood vessels in higher densities and with larger diameters, compared to other control conditions. Overall, the drug releasing system developed in this study will be broadly useful for regulating release rates of a wide array of protein drugs, and further enhance the quality of protein drug-based therapies. © 2013 Elsevier B.V. All rights reserved.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

PubMed

Jornot, L; Morris, M A; Petersen, H; Moix, I; Rochat, T

2002-01-01

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright 2001 John Wiley & Sons, Ltd.

The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

NASA Technical Reports Server (NTRS)

Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

1978-01-01

During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

Preparation of pH-sensitive zwitterionic nano micelles and drug controlled release for enhancing cellular uptake.

PubMed

Wu, Luyan; Ni, Caihua; Zhang, Liping; Shi, Gang

2016-01-01

Zwitterionic copolymers have exhibited high resistance to nonspecific protein adsorption and have wide applications in drug delivery systems. Herein, a pH-responsive poly(Lysine-alt-N,N'-bis(acryloyl) diaminohexane) was synthesized through the Michael addition polymerization between N, N'-bis(acryloyl) diaminohexane and lysine. Subsequently, nano micelles (NMs) were formed by self-assembly of the copolymer in an aqueous solution. The NMs showed a slightly negative charge in blood environment, but a positively charged surface in extracellular pH of tumor. This feature could be used to enhance permeability and retention effect, and reinforce tumor cell uptake. Vitro release studies revealed that the release of DOX from the DOX-loaded NMs was evidently faster at pH 5.0 than at pH 7.4. MTT assays revealed that NMs were nontoxic. Thus, these smart NMs were feasible candidates and could be potentially used in cancer chemotherapy.

Application of enhanced electronegative multimodal chromatography as the primary capture step for immunoglobulin G purification.

PubMed

Wang, Yanli; Chen, Quan; Xian, Mo; Nian, Rui; Xu, Fei

2018-06-01

In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.

Encapsulation of basic fibroblast growth factor by polyelectrolyte multilayer microcapsules and its controlled release for enhancing cell proliferation.

PubMed

She, Zhen; Wang, Chunxia; Li, Jun; Sukhorukov, Gleb B; Antipina, Maria N

2012-07-09

Basic fibroblast growth factor (FGF2) is an important protein for cellular activity and highly vulnerable to environmental conditions. FGF2 protected by heparin and bovine serum albumin was loaded into the microcapsules by a coprecipitation-based layer-by-layer encapsulation method. Low cytotoxic and biodegradable polyelectrolytes dextran sulfate and poly-L-arginine were used for capsule shell assembly. The shell thickness-dependent encapsulation efficiency was measured by enzyme-linked immunosorbent assay. A maximum encapsulation efficiency of 42% could be achieved by microcapsules with a shell thickness of 14 layers. The effects of microcapsule concentration and shell thickness on cytotoxicity, FGF2 release kinetics, and L929 cell proliferation were evaluated in vitro. The advantage of using microcapsules as the carrier for FGF2 controlled release for enhancing L929 cell proliferation was analyzed.

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study. PMID:22272336

BMP-2 Derived Peptide and Dexamethasone Incorporated Mesoporous Silica Nanoparticles for Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells.

PubMed

Zhou, Xiaojun; Feng, Wei; Qiu, Kexin; Chen, Liang; Wang, Weizhong; Nie, Wei; Mo, Xiumei; He, Chuanglong

2015-07-29

Bone morphogenetic protein-2 (BMP-2), a growth factor that induces osteoblast differentiation and promotes bone regeneration, has been extensively investigated in bone tissue engineering. The peptides of bioactive domains, corresponding to residues 73-92 of BMP-2 become an alternative to reduce adverse side effects caused by the use of high doses of BMP-2 protein. In this study, BMP-2 peptide functionalized mesoporous silica nanoparticles (MSNs-pep) were synthesized by covalently grafting BMP-2 peptide on the surface of nanoparticles via an aminosilane linker, and dexamethasone (DEX) was then loaded into the channel of MSNs to construct nanoparticulate osteogenic delivery systems (DEX@MSNs-pep). The in vitro cell viability of MSNs-pep was tested with bone mesenchymal stem cells (BMSCs) exposure to different particle concentrations, revealing that the functionalized MSNs had better cytocompatibility than their bare counterparts, and the cellular uptake efficiency of MSNs-pep was remarkably larger than that of bare MSNs. The in vitro results also show that the MSNs-pep promoted osteogenic differentiation of BMSCs in terms of the levels of alkaline phosphatase (ALP) activity, calcium deposition, and expression of bone-related protein. Moreover, the osteogenic differentiation of BMSCs can be further enhanced by incorporating of DEX into MSNs-pep. After intramuscular implantation in rats for 3 weeks, the computed tomography (CT) images and histological examination indicate that this nanoparticulate osteogenic delivery system induces effective osteoblast differentiation and bone regeneration in vivo. Collectively, the BMP-2 peptide and DEX incorporated MSNs can act synergistically to enhance osteogenic differentiation of BMSCs, which have potential applications in bone tissue engineering.

Effects of polymer graft properties on protein adsorption and transport in ion exchange chromatography: a multiscale modeling study.

PubMed

Basconi, Joseph E; Carta, Giorgio; Shirts, Michael R

2015-04-14

Multiscale simulation is used to study the adsorption of lysozyme onto ion exchangers obtained by grafting charged polymers into a porous matrix, in systems with various polymer properties and strengths of electrostatic interaction. Molecular dynamics simulations show that protein partitioning into the polymer-filled pore space increases with the overall charge content of the polymers, while the diffusivity in the pore space decreases. However, the combination of greatly increased partitioning and modestly decreased diffusion results in macroscopic transport rates that increase as a function of charge content, as the large concentration driving force due to enhanced pore space partitioning outweighs the reduction in the pore space diffusivity. Matrices having greater charge associated with the grafted polymers also exhibit more diffuse intraparticle concentration profiles during transient adsorption. In systems with a high charge content per polymer and a low protein loading, the polymers preferentially partition toward the surface due to favorable interactions with the surface-bound protein. These results demonstrate the potential of multiscale modeling to illuminate qualitative trends between molecular properties and the adsorption equilibria and kinetic properties observable on macroscopic scales.

Kainate receptor-mediated depression of glutamatergic transmission involving protein kinase A in the lateral amygdala.

PubMed

Negrete-Díaz, José Vicente; Duque-Feria, Paloma; Andrade-Talavera, Yuniesky; Carrión, Miriam; Flores, Gonzalo; Rodríguez-Moreno, Antonio

2012-04-01

Kainate receptors (KARs) have been described as modulators of synaptic transmission at different synapses. However, this role of KARs has not been well characterized in the amygdala. We have explored the effect of kainate receptor activation at the synapse established between fibers originating at medial geniculate nucleus and the principal cells in the lateral amygdala. We have observed an inhibition of evoked excitatory postsynaptic currents (eEPSCs) amplitude after a brief application of KARs agonists KA and ATPA. Paired-pulse recordings showed a clear pair pulse facilitation that was enhanced after KA or ATPA application. When postsynaptic cells were loaded with BAPTA, the depression of eEPSC amplitude observed after the perfusion of KAR agonists was not prevented. We have also observed that the inhibition of the eEPSCs by KARs agonists was prevented by protein kinase A but not by protein kinase C inhibitors. Taken together our results indicate that KARs present at this synapse are pre-synaptic and their activation mediate the inhibition of glutamate release through a mechanism that involves the activation of protein kinase A. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

A Study of BMP-2-Loaded Bipotential Electrolytic Complex around a Biphasic Calcium Phosphate-Derived (BCP) Scaffold for Repair of Large Segmental Bone Defect

PubMed Central

Paul, Kallyanashis; Padalhin, Andrew R.; Linh, Nguyen Thuy Ba; Kim, Boram; Sarkar, Swapan Kumar; Lee, Byong Taek

2016-01-01

A bipotential polyelectrolyte complex with biphasic calcium phosphate (BCP) powder dispersion provides an excellent option for protein adsorption and cell attachment and can facilitate enhanced bone regeneration. Application of the bipotential polyelectrolyte complex embedded in a spongy scaffold for faster healing of large segmental bone defects (LSBD) can be a promising endeavor in tissue engineering application. In the present study, a hollow scaffold suitable for segmental long bone replacement was fabricated by the sponge replica method applying the microwave sintering process. The fabricated scaffold was coated with calcium alginate at the shell surface, and genipin-crosslinked chitosan with biphasic calcium phosphate (BCP) dispersion was loaded at the central hollow core. The chitosan core was subsequently loaded with BMP-2. The electrolytic complex was characterized using SEM, porosity measurement, FTIR spectroscopy and BMP-2 release for 30 days. In vitro studies such as MTT, live/dead, cell proliferation and cell differentiation were performed. The scaffold was implanted into a 12 mm critical size defect of a rabbit radius. The efficacy of this complex is evaluated through an in vivo study, one and two month post implantation. BV/TV ratio for BMP-2 loaded sample was (42±1.76) higher compared with hollow BCP scaffold (32±0.225). PMID:27711142

Biodegradable fibre scaffolds incorporating water-soluble drugs and proteins.

PubMed

Ma, J; Meng, J; Simonet, M; Stingelin, N; Peijs, T; Sukhorukov, G B

2015-07-01

A new type of biodegradable drug-loaded fibre scaffold has been successfully produced for the benefit of water-soluble drugs and proteins. Model drug loaded calcium carbonate (CaCO3) microparticles incorporated into poly(lactic acid-co-glycolic acid) (PLGA) fibres were manufactured by co-precipitation of CaCO3 and the drug molecules, followed by electrospinning of a suspension of such drug-loaded microparticles in a PLGA solution. Rhodamine 6G and bovine serum albumin were used as model drugs for our release study, representing small bioactive molecules and protein, respectively. A bead and string structure of fibres was achieved. The drug release was investigated with different drug loadings and in different pH release mediums. Results showed that a slow and sustained drug release was achieved in 40 days and the CaCO3 microparticles used as the second barrier restrained the initial burst release.

Light-triggered liposomal cargo delivery platform incorporating photosensitizers and gold nanoparticles for enhanced singlet oxygen generation and increased cytotoxicity.

PubMed

Kautzka, Zofia; Clement, Sandhya; Goldys, Ewa M; Deng, Wei

2017-01-01

We developed light-triggered liposomes incorporating 3-5 nm hydrophobic gold nanoparticles and Rose Bengal (RB), a well-known photosensitizer used for photodynamic therapy. Singlet oxygen generated by these liposomes with 532 nm light illumination was characterized for varying the molar ratio of lipids and gold nanoparticles while keeping the amount of RB constant. Gold nanoparticles were found to enhance the singlet oxygen generation rate, with a maximum enhancement factor of 1.75 obtained for the molar ratio of hydrogenated soy l-α-phosphatidylcholine:1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(hexanoylamine):gold of 57:5:17 compared with liposomes loaded with RB alone. The experimental results could be explained by the local electric field enhancement caused by gold nanoparticles. We further assessed cellular cytotoxicity of gold-loaded liposomes by encapsulating an antitumor drug, doxorubicin (Dox); such Dox-loaded liposomes were applied to human colorectal cancer cells (HCT116) and exposed to light. Gold-loaded liposomes containing RB and Dox where Dox release was triggered by light were found to exhibit higher cytotoxicity compared with the liposomes loaded with RB and Dox alone. Our results indicate that gold-loaded liposomes incorporating photosensitizers may serve as improved agents in photodynamic therapy and chemotherapy.

Light-triggered liposomal cargo delivery platform incorporating photosensitizers and gold nanoparticles for enhanced singlet oxygen generation and increased cytotoxicity

PubMed Central

Kautzka, Zofia; Clement, Sandhya; Goldys, Ewa M; Deng, Wei

2017-01-01

We developed light-triggered liposomes incorporating 3–5 nm hydrophobic gold nanoparticles and Rose Bengal (RB), a well-known photosensitizer used for photodynamic therapy. Singlet oxygen generated by these liposomes with 532 nm light illumination was characterized for varying the molar ratio of lipids and gold nanoparticles while keeping the amount of RB constant. Gold nanoparticles were found to enhance the singlet oxygen generation rate, with a maximum enhancement factor of 1.75 obtained for the molar ratio of hydrogenated soy l-α-phosphatidylcholine:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(hexanoylamine):gold of 57:5:17 compared with liposomes loaded with RB alone. The experimental results could be explained by the local electric field enhancement caused by gold nanoparticles. We further assessed cellular cytotoxicity of gold-loaded liposomes by encapsulating an antitumor drug, doxorubicin (Dox); such Dox-loaded liposomes were applied to human colorectal cancer cells (HCT116) and exposed to light. Gold-loaded liposomes containing RB and Dox where Dox release was triggered by light were found to exhibit higher cytotoxicity compared with the liposomes loaded with RB and Dox alone. Our results indicate that gold-loaded liposomes incorporating photosensitizers may serve as improved agents in photodynamic therapy and chemotherapy. PMID:28203076

Translocator protein ligand-PLGA conjugated nanoparticles for 5-fluorouracil delivery to glioma cancer cells.

PubMed

Laquintana, Valentino; Denora, Nunzio; Lopalco, Antonio; Lopedota, Angela; Cutrignelli, Annalisa; Lasorsa, Francesco Massimo; Agostino, Giulia; Franco, Massimo

2014-03-03

Translocator protein 18 kDa (TSPO) is a promising target for molecular imaging and for targeted drug delivery to tumors overexpressing TSPO. In our previous work, new macromolecular conjugates with a high affinity and selectivity for TSPO were prepared by conjugating the biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) polymer with two potent and selective TSPO ligands, namely, compounds 1 and 2. Based on this, nanoparticle delivery systems (NPs), employing TSPO ligand-PLGA conjugated (PLGA-TSPO) polymers, were prepared. Furthermore, to evaluate the ability of the new NPs to be used as a drug delivery systems for anticancer therapy, PLGA-TSPO NPs were loaded with 5-fluorouracil (5-FU), chosen as a model hydrophilic anticancer drug. The main goal of this work was to investigate the synergistic potential of using NP conjugates PLGA-TSPO, TSPO ligands being pro-apoptotic agents, to simultaneously deliver a cytotoxic anticancer drug. To better highlight the occurrence of synergistic effects, dual drug loaded PLGA NPs (PLGA NPs/5-FU/1) and dual drug loaded PLGA-TSPO NPs (PLGA-TSPO NPs/5-FU/1), with 5-FU and TSPO ligand 1 physically incorporated together, were also prepared and characterized. The particle size and size distribution, surface morphology, and drug encapsulation efficiency, as well as the drug release kinetics, were investigated. In vitro cytotoxicity studies were carried out on C6 glioma cells overexpressing TSPO, and to evaluate the potential uptake of these nanoparticulate systems, the internalization of fluorescent labeled PLGA-TSPO NPs (FITC-PLGA-TSPO NPs) was also investigated by fluorescence microscopy. Results demonstrated that PLGA-TSPO NPs/5-FU and dual drug loaded PLGA NPs/5-FU/1 and PLGA-TSPO NPs/5-FU/1 could significantly enhance toxicity against human cancer cells due to the synergistic effect of the TSPO ligand 1 with the anticancer drug 5-FU.

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres*

PubMed Central

Jaramillo-Lambert, Aimee; Hao, Jing; Xiao, Haijie; Li, Yongming; Han, Zhiyong; Zhu, Wenge

2013-01-01

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G1 phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G1 phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres. PMID:23184928

Functionalization of alkyne-terminated thermally hydrocarbonized porous silicon nanoparticles with targeting peptides and antifouling polymers: effect on the human plasma protein adsorption.

PubMed

Wang, Chang-Fang; Mäkilä, Ermei M; Bonduelle, Colin; Rytkönen, Jussi; Raula, Janne; Almeida, Sérgio; Närvänen, Ale; Salonen, Jarno J; Lecommandoux, Sebastien; Hirvonen, Jouni T; Santos, Hélder A

2015-01-28

Porous silicon (PSi) nanomaterials combine a high drug loading capacity and tunable surface chemistry with various surface modifications to meet the requirements for biomedical applications. In this work, alkyne-terminated thermally hydrocarbonized porous silicon (THCPSi) nanoparticles were fabricated and postmodified using five bioactive molecules (targeting peptides and antifouling polymers) via a single-step click chemistry to modulate the bioactivity of the THCPSi nanoparticles, such as enhancing the cellular uptake and reducing the plasma protein association. The size of the nanoparticles after modification was increased from 176 to 180-220 nm. Dextran 40 kDa modified THCPSi nanoparticles showed the highest stability in aqueous buffer. Both peptide- and polymer-functionalized THCPSi nanoparticles showed an extensive cellular uptake which was dependent on the functionalized moieties presented on the surface of the nanoparticles. The plasma protein adsorption study showed that the surface modification with different peptides or polymers induced different protein association profiles. Dextran 40 kDa functionalized THCPSi nanoparticles presented the least protein association. Overall, these results demonstrate that the "click" conjugation of the biomolecules onto the alkyne-terminated THCPSi nanoparticles is a versatile and simple approach to modulate the surface chemistry, which has high potential for biomedical applications.

Diffusion-Limited Cargo Loading of an Engineered Protein Container.

PubMed

Zschoche, Reinhard; Hilvert, Donald

2015-12-30

The engineered bacterial nanocompartment AaLS-13 is a promising artificial encapsulation system that exploits electrostatic interactions for cargo loading. In order to study its ability to take up and retain guests, a pair of fluorescent proteins was developed which allows spectroscopic determination of the extent of encapsulation by Förster resonance energy transfer (FRET). The encapsulation process is generally complete within a second, suggesting low energetic barriers for proteins to cross the capsid shell. Formation of intermediate aggregates upon mixing host and guest in vitro complicates capsid loading at low ionic strength, but can be sidestepped by increasing salt concentrations or diluting the components. Encapsulation of guests is completely reversible, and the position of the equilibrium is easily tuned by varying the ionic strength. These results, which challenge the notion that AaLS-13 is a continuous rigid shell, provide valuable information about cargo loading that will guide ongoing efforts to engineer functional host-guest complexes. Moreover, it should be possible to adapt the protein FRET pair described in this report to characterize functional capsid-cargo complexes generated by other encapsulation systems.

Functional and in vitro gastric digestibility of the whey protein hydrogel loaded with nanostructured lipid carriers and gelled via citric acid-mediated crosslinking.

PubMed

Hashemi, Behnaz; Madadlou, Ashkan; Salami, Maryam

2017-12-15

Nanostructured lipid carriers (NLCs) with mean size of 347nm were fabricated and added into a heat-denatured whey protein solution. The subsequent crosslinking of proteins by citric acid or CaCl 2 resulted in the formation of cold-set hydrogels. Fourier transform infrared spectroscopy (FTIR) proposed formation of more hydrogen bonds in gel due to NLC loading or citric acid-mediated gelation. It was also found based on FITR spectroscopy that citric acid crosslinking disordered whey proteins. Scanning electron microscopy (SEM) imaging showed a non-porous and finely meshed microstructure for the crosslinked gels compared to non-crosslinked counterparts. Crosslinking also increased the firmness and water-holding capacity of gels. In pepsin-free fluid, a strong correlation existed between reduction in gel swellability and digestibility over periods up to 60min due to NLC loading and citric acid gelation. However, in peptic fluid, NLC loading and citric acid crosslinking brought about much higher decrease in digestibility than swellability. Copyright © 2017 Elsevier Ltd. All rights reserved.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies.

PubMed

Kelley, Brian D; Tobler, Scott A; Brown, Paul; Coffman, Jonathan L; Godavarti, Ranga; Iskra, Timothy; Switzer, Mary; Vunnum, Suresh

2008-10-15

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved. (c) 2008 Wiley Periodicals, Inc.

Standardization of formulations for the acute amino acid depletion and loading tests.

PubMed

Badawy, Abdulla A-B; Dougherty, Donald M

2015-04-01

The acute tryptophan depletion and loading and the acute tyrosine plus phenylalanine depletion tests are powerful tools for studying the roles of cerebral monoamines in behaviour and symptoms related to various disorders. The tests use either amino acid mixtures or proteins. Current amino acid mixtures lack specificity in humans, but not in rodents, because of the faster disposal of branched-chain amino acids (BCAAs) by the latter. The high content of BCAA (30-60%) is responsible for the poor specificity in humans and we recommend, in a 50g dose, a control formulation with a lowered BCAA content (18%) as a common control for the above tests. With protein-based formulations, α-lactalbumin is specific for acute tryptophan loading, whereas gelatine is only partially effective for acute tryptophan depletion. We recommend the use of the whey protein fraction glycomacropeptide as an alternative protein. Its BCAA content is ideal for specificity and the absence of tryptophan, tyrosine and phenylalanine render it suitable as a template for seven formulations (separate and combined depletion or loading and a truly balanced control). We invite the research community to participate in standardization of the depletion and loading methodologies by using our recommended amino acid formulation and developing those based on glycomacropeptide. © The Author(s) 2015.

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

PubMed Central

Stalder, Lukas; Heusermann, Wolf; Sokol, Lena; Trojer, Dominic; Wirz, Joel; Hean, Justin; Fritzsche, Anja; Aeschimann, Florian; Pfanzagl, Vera; Basselet, Pascal; Weiler, Jan; Hintersteiner, Martin; Morrissey, David V; Meisner-Kober, Nicole C

2013-01-01

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing. PMID:23511973

Enhanced colon cancer chemoprevention of curcumin by nanoencapsulation with whey protein.

PubMed

Jayaprakasha, Guddadarangavvanahally K; Chidambara Murthy, Kotamballi N; Patil, Bhimanagouda S

2016-10-15

To improve bioavailability and enhance colon cancer prevention ability of curcumin, whey protein was used to nanoencapsulate at three different ratios such as 70:30, 50:50 and 35:65 for the first time. The drug loading, entrapment efficiency and structural changes of curcumin was confirmed by quantitative NMR spectroscopy. The nanoparticles prepared using the three ratios had an average diameters of 236.5±8.8, 212±3.4, and 187±11.4nm, as well as zeta (ζ) potentials of -13.1,-9.26, and -4.63mV, respectively, at pH 7.0. The cytotoxicity assay was performed for human colon and prostate cancer (SW480 and LNCap) by MTT assay and results showed significantly higher cytotoxicity of nanoencapsulated curcumin (NEC) (equivalent to 30.91, 20.70 and 16.86µM of NEC-1, 2 and 3 respectively), as compared to plain curcumin at 50µM after 72h of treatment. Cytotoxicity was also confirmed by microscopy of treated cells stained with acridine orange and propidium iodide. The cells treated with 50µM of curcumin, 30.91µM (NEC-1), 20.70µM (NEC-2) and 16.86µM (NEC-3) showed enhanced activation of p53 and elevated bax/Bcl2 expression (NEC-3), increased cytochrome-c in cytosol (NEC-2) confirming the enhanced cytotoxicity. To confirm the increased bioavailability, the intracellular curcumin was measured using fluorescence intensity. The fluorescent signal for intracellular curcumin was increased by 12, 30, and 21% for NEC-1, NEC-2, and NEC-3 respectively as compared to plain curcumin at 4h. Based on these results, we conclude that nanoencapsulated curcumin with whey protein will have potential to be considered for clinical applications for future studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery

PubMed Central

Knock, Greg A.; Snetkov, Vladimir A.; Shaifta, Yasin; Drndarski, Svetlana; Ward, Jeremy P.T.; Aaronson, Philip I.

2008-01-01

Aims We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca2+ sensitization and changes in intracellular Ca2+ concentration ([Ca2+]i). Methods and results Intra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC20) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca2+]i was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC20 phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca2+]i that accompany HPV were also inhibited by PP2. Conclusion Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca2+ sensitization, and [Ca2+]i responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV. PMID:18682436

Gold nanoparticle-DNA aptamer composites as a universal carrier for in vivo delivery of biologically functional proteins.

PubMed

Ryou, Sang-Mi; Yeom, Ji-Hyun; Kang, Hyo Jung; Won, Miae; Kim, Jin-Sik; Lee, Boeun; Seong, Maeng-Je; Ha, Nam-Chul; Bae, Jeehyeon; Lee, Kangseok

2014-12-28

Although the delivery of biologically functional protein(s) into mammalian cells could be of tremendous value to biomedical research, the development of such technology has been hindered by the lack of a safe and effective delivery method. Here, we present a simple, efficient, and versatile gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system, with nanoblock-like properties, that allows any recombinant protein to be loaded without additional modifications and delivered into mammalian living systems. AuNP-Apt-based protein delivery system was able to deliver various proteins into variety of cell types in vitro without showing cytotoxicity. This AuNP-Apt system was also effective for the local and systemic targeted delivery of proteins in vivo. A local injection of the AuNP-Apt loaded with the apoptosis-inducing BIM protein efficiently inhibited the growth of xenograft tumors in mice. Furthermore, an intravenous injection of AuNP-Apt loaded with both epidermal growth factor (EGF) and BIM resulted in the targeted delivery of BIM into a xenograft tumor derived from EGF receptor-overexpressing cancer cells with no detectable systemic toxicity. Our findings show that this system can serve as an innovative platform for the development of protein-based biomedical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Lipid-coated mannitol core microparticles for sustained release of protein.

PubMed

Wang, Bifeng; Friess, Wolfgang

2018-07-01

Parenteral sustained release systems for proteins which provide therapeutic levels over a longer period avoiding frequent administration, which preserve protein stability during manufacturing, storage and application and which are biodegradable and highly biocompatible in the body are intensively sought after. The aim of this study was to generate and study mannitol core microparticles loaded with a monoclonal antibody IgG1 and coated with lipid either hard fat or glyceryl stearate at different coating levels. The protein was stabilized with 22.5 mg/mL sucrose, 0.1% PS 80, 10 mM methionine in 10 mM His buffer pH 7.2 during the spray loading process. 30 g protein-loaded mannitol carrier microparticles were coated with 5 g, 10 g, 20 g and 30 g of lipid, respectively. Placing more lipid onto the protein-loaded microparticles reduced both burst and release rate, and the particles maintained their geometric form during the release test. The IgG1 release from microparticles covered with a hard fat layer extended up to 6 weeks. The IgG1 was released in its monomeric form and maintained its secondary structure as shown by FTIR. Incomplete release of IgG1 from glyceryl stearate-coated microparticles was observed, which may be due to the small pore sizes of the glyceryl stearate layer or a detrimental surfactant character of glyceryl stearate to protein. Hence, these hard fat-coated mannitol core microparticles have high potential for protein delivery. Copyright © 2018 Elsevier B.V. All rights reserved.

Diurnal Changes of Polysome Loading Track Sucrose Content in the Rosette of Wild-Type Arabidopsis and the Starchless pgm Mutant1[W][OA

PubMed Central

Pal, Sunil Kumar; Liput, Magdalena; Piques, Maria; Ishihara, Hirofumi; Obata, Toshihiro; Martins, Marina C.M.; Sulpice, Ronan; van Dongen, Joost T.; Fernie, Alisdair R.; Yadav, Umesh Prasad; Lunn, John E.; Usadel, Björn; Stitt, Mark

2013-01-01

Growth is driven by newly fixed carbon in the light, but at night it depends on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night, and low CO2 in Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) and in the starchless phosphoglucomutase (pgm) mutant. In Col-0, polysome loading was 60% to 70% in the light, 40% to 45% for much of the night, and less than 20% in an extended night, while in pgm, it fell to less than 25% early in the night. Quantification of ribosomal RNA species using quantitative reverse transcription-polymerase chain reaction revealed that polysome loading remained high for much of the night in the cytosol, was strongly light dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared with Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from 13CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modeling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in the previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon. PMID:23674104

Protein Phosphatase 2A Antagonizes ATM and ATR in a Cdk2- and Cdc7-Independent DNA Damage Checkpoint

PubMed Central

Petersen, Paris; Chou, Danny M.; You, Zhongsheng; Hunter, Tony; Walter, Johannes C.; Walter, Gernot

2006-01-01

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts. PMID:16479016

Preparation and Drug-Delivery Properties of HKUST-1/GO Hybrid.

PubMed

Sun, Ke Ke; Li, Ling; He, Yu Qi; Fan, Lu; Wu, Ya Qi; Liu, Li

2016-01-01

A hybrid HKUST-1/GO composite was synthesized and its drug loading and drug release abilities were investigated. The adsorption of IBU (ibuprofen) onto the surface of HKUST-1/GO and HKUST-1 composites was compared, and it was found that the addition of GO enhanced both IBU loading and stability. The addition of GO also enhanced the specific surface area. Drug release experiments on IBU loaded HKUST-1 and HKUST-1/GO were conducted, and it was found that drug release of HKUST-1/GO was slower, which can be explained by the hydrogen bonding between GO and IBU. It can be concluded that the addition of GO not only enhances drug loading, but can also achieve a more desirable slow-release of the drug.

Pterostilbene, a Potent Analog of Resveratrol, a Therapeutic Agent in Prostate Cancer: Epigenetic Mechanisms of Action

DTIC Science & Technology

2015-10-01

protein was loaded in 10-15% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in 5% nonfat dry milk ...epigenetic reader, metastasis-associated protein 1 (MTA1) which is a part of nucleosome remodeling and deacetylation (NuRD) co-repressor complex. MTA1...inhibitor cocktail (ThermoFisher Scientific). Samples containing 70 μg of protein were loaded in 10-12% SDS-PAGE and transferred onto polyvinylidene

Buckling analysis of orthotropic protein microtubules under axial and radial compression based on couple stress theory.

PubMed

Beni, Yaghoub Tadi; Zeverdejani, M Karimi; Mehralian, Fahimeh

2017-10-01

Protein microtubules (MTs) are one of the important intercellular components and have a vital role in the stability and strength of the cells. Due to applied external loads, protein microtubules may be involved buckling phenomenon. Due to impact of protein microtubules in cell reactions, it is important to determine their critical buckling load. Considering nature of protein microtubules, various parameters are effective on microtubules buckling. The small size of microtubules and also lack of uniformity of MTs properties in different directions caused the necessity of accuracy in the analysis of these bio-structure. In fact, microtubules must be considered as a size dependent cylinder, which behave as an orthotropic material. Hence, in the present work using first-order shear deformation model (FSDT), the buckling equations of anisotropic MTs are derived based on new modified couple stress theory (NMCST). After solving the stability equations, the influences of various parameters are measured on the MTs critical buckling load. Copyright © 2017 Elsevier Inc. All rights reserved.

Influence of dietary protein on postprandial blood glucose levels in individuals with Type 1 diabetes mellitus using intensive insulin therapy.

PubMed

Paterson, M A; Smart, C E M; Lopez, P E; McElduff, P; Attia, J; Morbey, C; King, B R

2016-05-01

To determine the effects of protein alone (independent of fat and carbohydrate) on postprandial glycaemia in individuals with Type 1 diabetes mellitus using intensive insulin therapy. Participants with Type 1 diabetes mellitus aged 7-40 years consumed six 150 ml whey isolate protein drinks [0 g (control), 12.5, 25, 50, 75 and 100] and two 150 ml glucose drinks (10 and 20 g) without insulin, in randomized order over 8 days, 4 h after the evening meal. Continuous glucose monitoring was used to assess postprandial glycaemia. Data were collected from 27 participants. Protein loads of 12.5 and 50 g did not result in significant postprandial glycaemic excursions compared with control (water) throughout the 300 min study period (P > 0.05). Protein loads of 75 and 100 g resulted in lower glycaemic excursions than control in the 60-120 min postprandial interval, but higher excursions in the 180-300 min interval. In comparison with 20 g glucose, the large protein loads resulted in significantly delayed and sustained glucose excursions, commencing at 180 min and continuing to 5 h. Seventy-five grams or more of protein alone significantly increases postprandial glycaemia from 3 to 5 h in people with Type 1 diabetes mellitus using intensive insulin therapy. The glycaemic profiles resulting from high protein loads differ significantly from the excursion from glucose in terms of time to peak glucose and duration of the glycaemic excursion. This research supports recommendations for insulin dosing for large amounts of protein. © 2015 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING

PubMed Central

Zhang, Lu; Mo, Jinyao; Swanson, Karen V.; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M.; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A.; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom A; Shu, Hong-Bing; Duncan, Joseph A.; Ting, Jenny P-Y.

2014-01-01

SUMMARY Stimulator of interferon genes (STING, also named MITA, MYPS or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich repeat containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses. NLRC3 associated with both STING and TBK1, and impeded STING-TBK1 interaction and downstream type I interferon production. Using purified recombinant proteins NLRC3 was found to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3−/− mice exhibited enhanced innate immunity, reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus and c-di-GMP PMID:24560620

Hepatic fibrosis and carcinogenesis in α1-antitrypsin deficiency: a prototype for chronic tissue damage in gain-of-function disorders.

PubMed

Perlmutter, David H; Silverman, Gary A

2011-03-01

In α1-antitrypsin (AT) deficiency, a point mutation renders a hepatic secretory glycoprotein prone to misfolding and polymerization. The mutant protein accumulates in the endoplasmic reticulum of liver cells and causes hepatic fibrosis and hepatocellular carcinoma by a gain-of-function mechanism. Genetic and/or environmental modifiers determine whether an affected homozygote is susceptible to hepatic fibrosis/carcinoma. Two types of proteostasis mechanisms for such modifiers have been postulated: variation in the function of intracellular degradative mechanisms and/or variation in the signal transduction pathways that are activated to protect the cell from protein mislocalization and/or aggregation. In recent studies we found that carbamazepine, a drug that has been used safely as an anticonvulsant and mood stabilizer, reduces the hepatic load of mutant AT and hepatic fibrosis in a mouse model by enhancing autophagic disposal of this mutant protein. These results provide evidence that pharmacological manipulation of endogenous proteostasis mechanisms is an appealing strategy for chemoprophylaxis in disorders involving gain-of-function mechanisms.

Self assembled dual responsive micelles stabilized with protein for co-delivery of drug and siRNA in cancer therapy.

PubMed

Aji Alex, M R; Nehate, Chetan; Veeranarayanan, Srivani; Kumar, D Sakthi; Kulshreshtha, Ritu; Koul, Veena

2017-07-01

Design of safe and efficient vehicles for the combinatorial delivery of drugs and genetic agents is an emerging requisite for achieving enhanced therapeutic effect in cancer. Even though several nanoplatforms have been explored for the co-delivery of drugs and genetic materials the translation of these systems to clinical phase is still a challenge, mainly due to tedious synthesis procedures, lack of serum stability, inefficient scalability etc. Here in, we report development of reduction and pH sensitive polymeric graft of low molecular weight poly (styrene -alt -maleic anhydride) and evaluation of its efficacy in co-delivering drug and siRNA. The polymer was modified with suitable components, which could help in overcoming various systemic and cellular barriers for successful co-delivery of drugs and nucleic acids to cancer cells, using simple chemical reactions. The polymeric derivative could easily self assemble in water to form smooth, spherical micellar structures, indicating their scalability. Doxorubicin and PLK-1 siRNA were selected as model drug and nucleic acid, respectively. Doxorubicin could be loaded in the self assembling micelles with an optimum loading content of ∼8.6% w/w and efficient siRNA complexation was achieved with polymer/siRNA weight ratios >40. The polyplexes were stabilized in physiological saline by coating with bovine serum albumin (BSA). Stable drug loaded nanoplexes, for clinical administration, could be easily formulated by gently dispersing them in physiological saline containing appropriate amount of albumin. Drug release from the nanoplexes was significantly enhanced at low pH (5) and in the presence of 10 mM glutathione (GSH) showing their dual stimuli sensitive nature. In vitro cell proliferation assay and in vivo tumor regression study have shown synergistic effect of the drug loaded nanoplexes in inhibiting cancer cell proliferation. Facile synthesis steps, scalability and ease of formulation depict excellent clinical translation potential of the proposed nanosystem. Copyright © 2017 Elsevier Ltd. All rights reserved.

Degradable polymeric nano-films and particles as delivery platforms for vaccines and immunotherapeutics

NASA Astrophysics Data System (ADS)

Su, Xingfang

Degradable polymeric materials provide opportunities for the development of improved vaccines and immunotherapies by acting as platforms that facilitate the delivery of molecules to appropriate tissue and cellular locations to achieve therapeutic outcomes. To this end, we have designed and characterized nano-films and particles employing a hydrolytically degradable polymer for the delivery of vaccine antigens and immunotherapeutics. We first describe protein- and oligonucleotide-loaded layer-by-layer (LbL)-assembled multilayer thin films constructed based on electrostatic interactions between a cationic poly(beta-amino ester) (PBAE, denoted Poly-1) with a model protein antigen, ovalbumin (OVA), and/or immunostimulatory CpG oligonucleotides for transcutaneous delivery. Linear growth of nanoscale Poly-I/OVA bilayers was observed. Dried OVA protein-loaded films rapidly deconstructed when rehydrated in saline solutions, releasing OVA as non-aggregated/non-degraded protein, suggesting that the structure of biomolecules integrated into these multilayer films are preserved during release. Using confocal fluorescence microscopy and an in vivo murine ear skin model, we demonstrated delivery of OVA from LbL films into barrier-disrupted skin, uptake of the protein by skin-resident antigen-presenting cells (Langerhans cells), and transport of the antigen to the skin-draining lymph nodes. Dual incorporation of OVA and CpG oligonucleotides into the nanolayers of LbL films enabled dual release of the antigen and adjuvant with distinct kinetics for each component; OVA was rapidly released while CpG was released in a relatively sustained manner. Applied as skin patches, these films delivered OVA and CpG to Langerhans Cells in the skin. To our knowledge, this is the first demonstration of LbL films applied for the delivery of biomolecules into skin. This approach provides a new route for storage of vaccines and other immunotherapeutics in a solid-state thin film for subsequent delivery into the immunologically-rich milieu of the skin. In parallel, we also developed biodegradable core-shell nanoparticles with a PBAE core enveloped by a phospholipid bilayer shell for cytosolic delivery, with a view towards delivery of messenger RNA (mRNA)-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. mRNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of ˜30%. Particles also promoted cytosolic uptake of co-delivered anti-tumor toxins in tumor cells resulting in synergistic killing, demonstrating potential for cancer therapy. In vivo, particles loaded with mRNA administered intranasally or intratracheally in mice led to the enhanced expression of the reporter protein luciferase compared to naked mRNA This system may thus be promising for noninvasive delivery of mRNA-based vaccines. (Copies available exclusively from MIT Libraries, libraries.mit.edu/docs - docs mit.edu)

Cyclodextrin-PEG conjugate-wrapped magnetic ferrite nanoparticles for enhanced drug loading and release

NASA Astrophysics Data System (ADS)

Enoch, Israel V. M. V.; Ramasamy, Sivaraj; Mohiyuddin, Shanid; Gopinath, Packirisamy; Manoharan, R.

2018-05-01

Magnetic nanoparticles are envisaged to overcome the impediments in the methods of targeted drug delivery and hence cure cancer effectively. We report herein, manganese ferrite nanoparticles, coated with β-cyclodextrin-modified polyethylene glycol as a carrier for the drug, camptothecin. The particles are of the size of 100 nm and they show superparamagnetic behaviour. The saturation magnetization does not get diminished on polymer coverage of the nanoparticles. The β-cyclodextrin-polyethylene glycol conjugates are characterized using NMR and mass spectrometric techniques. By coating the magnetic nanoparticles with the cyclodextrin-tethered polymer, the drug-loading capacity is enhanced and the observed release of the drug is slow and sustained. The cell viability of HEK293 and HCT15 cells is evaluated and the cytotoxicity is enhanced when the drug is loaded in the polymer-coated magnetic nanoparticles. The noncovalent-binding based and enhanced drug loading on the nanoparticles and the sustained release make the nanocarrier a promising agent for carrying the payload to the target.

Sodium chloride promotes tissue inflammation via osmotic stimuli in subtotal-nephrectomized mice.

PubMed

Sakata, Fumiko; Ito, Yasuhiko; Mizuno, Masashi; Sawai, Akiho; Suzuki, Yasuhiro; Tomita, Takako; Tawada, Mitsuhiro; Tanaka, Akio; Hirayama, Akiyoshi; Sagara, Akihiro; Wada, Takashi; Maruyama, Shoichi; Soga, Tomoyoshi; Matsuo, Seiichi; Imai, Enyu; Takei, Yoshifumi

2017-04-01

Chronic inflammation, which is often associated with high all-cause and cardiovascular mortality, is prevalent in patients with renal failure; however, the precise mechanisms remain unclear. High-salt intake was reported to induce lymphangiogenesis and autoimmune diseases via osmotic stimuli with accumulation of sodium or chloride. In addition, sodium was recently reported to be stored in the extremities of dialysis patients. We studied the effects and mechanisms of high salt loading on tissue and systemic inflammation in subtotal-nephrectomized mice (5/6Nx) and in cultured cells. Macrophage infiltration in the peritoneal wall (P<0.001), heart (P<0.05) and para-aortic tissues (P<0.001) was significantly higher in 5/6Nx with salt loading (5/6Nx/NaCl) than in 5/6Nx without salt loading (5/6Nx/Water); however, there were no significant differences in blood pressure and renal function between the groups. Tissue interleukin-6, monocyte chemotactic protein-1 (MCP-1), serum- and glucocorticoid-inducible kinase 1 (Sgk1) and tonicity-responsive enhancer binding protein (TonEBP) mRNA were significantly elevated in the peritoneal wall and heart with 5/6Nx/NaCl when compared with 5/6Nx/Water. Sodium was stored in the abdominal wall, exerting high-osmotic conditions. Reversal of salt loading reduced macrophage infiltration associated with decreased TonEBP in 5/6Nx/NaCl. Macrophage infiltration associated with fibrosis induced by salt loading was decreased in the 5/6Nx/NaCl/CC chemokine receptor 2 (CCR2, receptor of MCP-1)-deficient mice when compared with 5/6Nx/NaCl/Wild mice, suggesting that CCR2 is required for macrophage infiltration in 5/6Nx with NaCl loading. In cultured mesothelial cells and cardiomyocytes, culture media with high NaCl concentration induced MCP-1, Sgk1 and TonEBP mRNA, all of which were suppressed by TonEBP siRNA, indicating that both MCP-1 and Sgk1 are downstream of TonEBP. Our study indicates that high NaCl intake induces MCP-1 expression leading to macrophage infiltration via the TonEBP-MCP-1 pathway in 5/6Nx/NaCl mice, and that TonEBP has a central role in inflammation in patients with renal failure taking high salt.

Modulating Cellular Recombination Potential through Alterations in RecA Structure and Regulation

PubMed Central

Bakhlanova, Irina V.; Dudkina, Alexandra V.; Baitin, Dima M.; Knight, Kendall L.; Cox, Michael M.; Lanzov, Vladislav A.

2010-01-01

The wild type E. coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to 6 fold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to 4 fold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50 fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of Escherichia coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function – filament formation and the inherent DNA pairing activity of the formed filaments. PMID:21143322

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

PubMed Central

Carlson, Brian E.; Vigoreaux, Jim O.; Maughan, David W.

2014-01-01

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10−10 cm2 s−1) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10−7 cm2 s−1 (parvalbumin) to 0.20 × 10−7 cm2 s−1 (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand. PMID:24559981

Poly(acrylic acid) brushes pattern as a 3D functional biosensor surface for microchips

NASA Astrophysics Data System (ADS)

Wang, Yan-Mei; Cui, Yi; Cheng, Zhi-Qiang; Song, Lu-Sheng; Wang, Zhi-You; Han, Bao-Hang; Zhu, Jin-Song

2013-02-01

Poly(acrylic acid) (PAA) brushes, a novel three dimensional (3D) precursor layer of biosensor or protein microarrays, possess high protein loading level and low non-specific protein adsorption. In this article, we describe a simple and convenient way to fabricate 3D PAA brushes pattern by microcontact printing (μCP) and characterize it with FT-IR and optical microscopy. The carboxyl groups of PAA brushes can be applied to covalently immobilize protein for immunoassay. Thriving 3D space made by patterning PAA brushes thin film is available to enhance protein immobilization, which is confirmed by measuring model protein interaction between human immunoglobulin G (H-IgG) and goat anti-H-IgG (G-H-IgG) with fluorescence microscopy and surface plasmon resonance imaging (SPRi). As expected, the SPRi signals of H-IgG coating on 3D PAA brushes pattern and further measuring specific binding with G-H-IgG are all larger than that of 3D PAA brushes without pattern and 2D bare gold surface. We further revealed that this surface can be used for high-throughput screening and clinical diagnosis by label-free assaying of Hepatitis-B-Virus surface antibody (HBsAb) with Hepatitis-B-Virus surface antigen (HBsAg) concentration array chip. The linearity range for HBsAb assay is wider than that of conventional ELISA method.

Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

PubMed

Mascarenhas, Roshan; Pietrzak, Maciej; Smith, Ryan M; Webb, Amy; Wang, Danxin; Papp, Audrey C; Pinsonneault, Julia K; Seweryn, Michal; Rempala, Grzegorz; Sadee, Wolfgang

2015-01-01

mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

Anti-inflammatory effects of EMD in the presence of biomechanical loading and interleukin-1β in vitro.

PubMed

Nokhbehsaim, Marjan; Deschner, Birgit; Winter, Jochen; Bourauel, Christoph; Jäger, Andreas; Jepsen, Søren; Deschner, James

2012-02-01

Enamel matrix derivative (EMD) used to promote periodontal regeneration has been shown to exert anti-inflammatory effects. This in vitro study was performed to investigate if the anti-inflammatory actions of EMD are modulated by the local cellular environment, such as inflammation or occlusal, i.e., biomechanical, loading. Human periodontal ligament cells were seeded on BioFlex plates and incubated with EMD under normal, inflammatory, and biomechanical loading conditions for 1 and 6 days. In order to mimic inflammatory and biomechanical loading conditions in vitro, cells were stimulated with interleukin (IL)-1β and exposed to dynamic tensile strain, respectively. The gene expression of IL-1β, IL-1 receptor antagonist (IL-1RN), IL-6, IL-8, IL-10, and cyclooxygenase (COX)-2 was analyzed by real-time RT-PCR and the IL-6 protein synthesis by enzyme-linked immunoassay. For statistical analysis, Student's t test, ANOVA, and post-hoc comparison tests were applied (p < 0.05). EMD downregulated significantly the expression of IL-1β and COX-2 at 1 day and of IL-6, IL-8, and COX-2 at 6 days in normal condition. In an inflammatory environment, the anti-inflammatory actions of EMD were significantly enhanced at 6 days. In the presence of low biomechanical loading, EMD caused a downregulation of IL-1β and IL-8, whereas high biomechanical loading significantly abrogated the anti-inflammatory effects of EMD at both days. Neither IL-1RN nor IL-10 was upregulated by EMD. These data suggest that high occlusal forces may abrogate anti-inflammatory effects of EMD and should, therefore, be avoided immediately after the application of EMD to achieve best healing results.

Development of implants composed of bioactive materials for bone repair

NASA Astrophysics Data System (ADS)

Xiao, Wei

The purpose of this Ph.D. research was to address the clinical need for synthetic bioactive materials to heal defects in non-loaded and loaded bone. Hollow hydroxyapatite (HA) microspheres created in a previous study were evaluated as a carrier for controlled release of bone morphogenetic protein-2 (BMP2) in bone regeneration. New bone formation in rat calvarial defects implanted with BMP2-loaded microspheres (43%) was significantly higher than microspheres without BMP2 (17%) at 6 weeks postimplantation. Then hollow HA microspheres with a carbonate-substituted composition were prepared to improve their resorption rate. Hollow HA microspheres with 12 wt. % of carbonate showed significantly higher new bone formation (73 +/- 8%) and lower residual HA (7 +/- 2%) than stoichiometric HA microspheres (59 +/- 2% new bone formation; 21 +/- 3% residual HA). The combination of carbonate-substituted hollow HA microspheres and clinically-safe doses of BMP2 could provide promising implants for healing non-loaded bone defects. Strong porous scaffolds of bioactive silicate (13-93) glass were designed with the aid of finite-element modeling, created by robocasting and evaluated for loaded bone repair. Scaffolds with a porosity gradient to mimic human cortical bone showed a compressive strength of 88 +/- 20 MPa, a flexural strength of 34 +/- 5 MPa and the ability to support bone infiltration in vivo. The addition of a biodegradable polylactic acid (PLA) layer to the external surface of these scaffolds increased their load-bearing capacity in four-point bending by 50% and dramatically enhanced their work of fracture, resulting in a "ductile" mechanical response. These bioactive glass-PLA composites, combining bioactivity, high strength, high work of fracture and an internal architecture conducive to bone infiltration, could provide optimal implants for structural bone repair.

Preparation, characterization and in vivo evaluation of a combination delivery system based on hyaluronic acid/jeffamine hydrogel loaded with PHBV/PLGA blend nanoparticles for prolonged delivery of Teriparatide.

PubMed

Bahari Javan, Nika; Montazeri, Hamed; Rezaie Shirmard, Leila; Jafary Omid, Nersi; Barbari, Ghullam Reza; Amini, Mohsen; Ghahremani, Mohammad Hossein; Rafiee-Tehrani, Morteza; Abedin Dorkoosh, Farid

2017-04-01

In the current study, biodegradable PHBV/PLGA blend nanoparticles (NPs) containing Teriparatide were loaded in hyaluronic acid/jeffamine (HA-JEF ED-600) hydrogel to prepare a combination delivery system (CDS) for prolonged delivery of Teriparatide. The principal purpose of the present study was to formulate an effective and prolonged Teriparatide delivery system in order to reduce the frequency of injection and thus enhance patient's compliance. Morphological properties, swelling behaviour, crosslinking efficiency and rheological characterization of HA-JEF ED-600 hydrogel were evaluated. The CDS was acquired by adding PHBV/PLGA NPs to HA-JEF ED-600 hydrogel simultaneously with crosslinking reaction. The percentage of NPs incorporation within the hydrogel as well as the loading capacity and morphology of Teriparatide loaded CDS were examined. Intrinsic fluorescence and circular dichroism spectroscopy proved that Teriparatide remains stable after processing. The release profile represented 63% Teriparatide release from CDS within 50days with lower burst release compared to NPs and hydrogel. MTT assay was conducted by using NIH3T3 cell line and no sign of reduction in cell viability was observed. Based on Miller and Tainter method, LD 50 of Teriparatide loaded CDS was 131.8mg/kg. In vivo studies demonstrated that Teriparatide loaded CDS could effectively increase serum calcium level after subcutaneous injection in mice. Favourable results in the current study introduced CDS as a promising candidate for controlled delivery of Teriparatide and pave the way for future investigations in the field of designing prolonged delivery systems for other peptides and proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation

PubMed Central

Weiffert, Tanja; Ní Mhurchú, Niamh; O’Connell, David; Linse, Sara

2016-01-01

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation. PMID:27812162

Effects of mechanical repetitive load on bone quality around implants in rat maxillae

PubMed Central

Uto, Yusuke; Nakano, Takayoshi; Ishimoto, Takuya; Inaba, Nao; Uchida, Yusuke; Sawase, Takashi

2017-01-01

Greater understanding and acceptance of the new concept “bone quality”, which was proposed by the National Institutes of Health and is based on bone cells and collagen fibers, are required. The novel protein Semaphorin3A (Sema3A) is associated with osteoprotection by regulating bone cells. The aims of this study were to investigate the effects of mechanical loads on Sema3A production and bone quality based on bone cells and collagen fibers around implants in rat maxillae. Grade IV-titanium threaded implants were placed at 4 weeks post-extraction in maxillary first molars. Implants received mechanical loads (10 N, 3 Hz for 1800 cycles, 2 days/week) for 5 weeks from 3 weeks post-implant placement to minimize the effects of wound healing processes by implant placement. Bone structures, bone mineral density (BMD), Sema3A production and bone quality based on bone cells and collagen fibers were analyzed using microcomputed tomography, histomorphometry, immunohistomorphometry, polarized light microscopy and birefringence measurement system inside of the first and second thread (designated as thread A and B, respectively), as mechanical stresses are concentrated and differently distributed on the first two threads from the implant neck. Mechanical load significantly increased BMD, but not bone volume around implants. Inside thread B, but not thread A, mechanical load significantly accelerated Sema3A production with increased number of osteoblasts and osteocytes, and enhanced production of both type I and III collagen. Moreover, mechanical load also significantly induced preferential alignment of collagen fibers in the lower flank of thread B. These data demonstrate that mechanical load has different effects on Sema3A production and bone quality based on bone cells and collagen fibers between the inside threads of A and B. Mechanical load-induced Sema3A production may be differentially regulated by the type of bone structure or distinct stress distribution, resulting in control of bone quality around implants in jaw bones. PMID:29244883

Effects of mechanical repetitive load on bone quality around implants in rat maxillae.

PubMed

Uto, Yusuke; Kuroshima, Shinichiro; Nakano, Takayoshi; Ishimoto, Takuya; Inaba, Nao; Uchida, Yusuke; Sawase, Takashi

2017-01-01

Greater understanding and acceptance of the new concept "bone quality", which was proposed by the National Institutes of Health and is based on bone cells and collagen fibers, are required. The novel protein Semaphorin3A (Sema3A) is associated with osteoprotection by regulating bone cells. The aims of this study were to investigate the effects of mechanical loads on Sema3A production and bone quality based on bone cells and collagen fibers around implants in rat maxillae. Grade IV-titanium threaded implants were placed at 4 weeks post-extraction in maxillary first molars. Implants received mechanical loads (10 N, 3 Hz for 1800 cycles, 2 days/week) for 5 weeks from 3 weeks post-implant placement to minimize the effects of wound healing processes by implant placement. Bone structures, bone mineral density (BMD), Sema3A production and bone quality based on bone cells and collagen fibers were analyzed using microcomputed tomography, histomorphometry, immunohistomorphometry, polarized light microscopy and birefringence measurement system inside of the first and second thread (designated as thread A and B, respectively), as mechanical stresses are concentrated and differently distributed on the first two threads from the implant neck. Mechanical load significantly increased BMD, but not bone volume around implants. Inside thread B, but not thread A, mechanical load significantly accelerated Sema3A production with increased number of osteoblasts and osteocytes, and enhanced production of both type I and III collagen. Moreover, mechanical load also significantly induced preferential alignment of collagen fibers in the lower flank of thread B. These data demonstrate that mechanical load has different effects on Sema3A production and bone quality based on bone cells and collagen fibers between the inside threads of A and B. Mechanical load-induced Sema3A production may be differentially regulated by the type of bone structure or distinct stress distribution, resulting in control of bone quality around implants in jaw bones.

Light-triggered liposomal cargo delivery platform incorporating photosensitizers and gold nanoparticles for enhanced singlet oxygen generation and increased cytotoxicity

NASA Astrophysics Data System (ADS)

Kautzka, Zofia; Clement, Sandhya; Goldys, Ewa M.; Deng, Wei

2018-02-01

We developed light-triggered liposomes incorporating gold nanoparticles and Rose Bengal (RB), a well-known photosensitizer used for photodynamic therapy. Singlet oxygen generated by these liposomes with 532 nm light illumination was characterized by adjusting the molar ratio of lipids and gold nanoparticles while keeping the amount of RB constant. Gold nanoparticles were found to enhance the singlet oxygen generation rate, with a maximum enhancement factor of 1.75 obtained for the molar ratio of HSPC: PE-NH2: gold of 57:5:17 compared with liposomes loaded with RB alone. The experimental results could be explained by the local electric field enhancement caused by gold nanoparticles. We further assessed cellular cytotoxicity of these liposomes by encapsulating an antitumor drug, doxorubicin (Dox); such Dox loaded liposomes were applied to human colorectal cancer cells, HCT116, and exposed to light. Gold-loaded liposomes containing RB and Dox where Dox release was triggered by light were found to exhibit higher cytotoxicity, compared to the liposomes loaded with RB and Dox alone. Our results indicate that gold-loaded liposomes incorporating photosensitizers may have improved therapeutic efficacy in photodynamic therapy and chemotherapy.

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

PubMed

Johnstone, Daniel; Graham, Ross M; Trinder, Debbie; Delima, Roheeth D; Riveros, Carlos; Olynyk, John K; Scott, Rodney J; Moscato, Pablo; Milward, Elizabeth A

2012-04-11

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Allium sativum-derived allitridin inhibits Treg amplification in cytomegalovirus infection.

PubMed

Li, Ya-nan; Huang, Fei; Liu, Xing-lou; Shu, Sai-nan; Huang, Yong-jian; Cheng, Huan-ji; Fang, Feng

2013-03-01

This study investigated the effects of allitridin compound on murine cytomegalovirus (MCMV)-induced regulatory T cell (Treg; CD4(+) CD25(+) Foxp3(+) ) amplification in vivo and in vitro. One hundred twenty MCMV-infected mice were allocated at random into two groups for treatment with allitridin or placebo. Another 120 mock-infected mice were randomly allocated as controls for the allitridin treatment and placebo treatment groups. The mice were euthanized at various time points after infection (out to 120 days) to evaluate the effects of treatment on Treg presence and function, as well as MCMV infective load. Co-culture with mouse embryo fibroblasts (MEF) and MCMV was performed to evaluate allitridin-mediated Treg and anti-CMV effects. The maximum tolerance concentration (MTC) of allitridin was used to treat cells for 3 days. Changes in Foxp3 mRNA and protein levels, percentages of T cell subsets, and Treg-related cytokines (IL-10 and TGF-β) were measured. Allitridin treatment did not influence Foxp3 expression and Treg proportion in uninfected mice, but did down-regulate each in infected mice during the chronic infection period. Additionally, allitridin treatment reduced the MCMV load in salivary glands. MTC allitridin treatment of co-cultures partially blocked MCMV induction of Foxp3 mRNA and protein expression. In vitro treatment with allitridin also increased significantly the percentages of Tc1, Tc2, and Th1, reduced the secreted levels of IL-10 and TGF-β1, and significantly suppressed viral loads. In conclusion, allitridin can promote MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression. Therefore, allitridin may be useful as a therapeutic agent to enhance the specific cellular immune responses against CMV. Copyright © 2013 Wiley Periodicals, Inc.

Autophagic clearance of mitochondria in the kidney copes with metabolic acidosis.

PubMed

Namba, Tomoko; Takabatake, Yoshitsugu; Kimura, Tomonori; Takahashi, Atsushi; Yamamoto, Takeshi; Matsuda, Jun; Kitamura, Harumi; Niimura, Fumio; Matsusaka, Taiji; Iwatani, Hirotsugu; Matsui, Isao; Kaimori, Junya; Kioka, Hidetaka; Isaka, Yoshitaka; Rakugi, Hiromi

2014-10-01

Metabolic acidosis, a common complication of CKD, causes mitochondrial stress by undefined mechanisms. Selective autophagy of impaired mitochondria, called mitophagy, contributes toward maintaining cellular homeostasis in various settings. We hypothesized that mitophagy is involved in proximal tubular cell adaptations to chronic metabolic acidosis. In transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3), NH4Cl loading increased the number of GFP puncta exclusively in the proximal tubule. In vitro, culture in acidic medium produced similar results in proximal tubular cell lines stably expressing GFP-LC3 and facilitated the degradation of SQSTM1/p62 in wild-type cells, indicating enhanced autophagic flux. Upon acid loading, proximal tubule-specific autophagy-deficient (Atg5-deficient) mice displayed significantly reduced ammonium production and severe metabolic acidosis compared with wild-type mice. In vitro and in vivo, acid loading caused Atg5-deficient proximal tubular cells to exhibit reduced mitochondrial respiratory chain activity, reduced mitochondrial membrane potential, and fragmented morphology with marked swelling in mitochondria. GFP-LC3-tagged autophagosomes colocalized with ubiquitinated mitochondria in proximal tubular cells cultured in acidic medium, suggesting that metabolic acidosis induces mitophagy. Furthermore, restoration of Atg5-intact nuclei in Atg5-deficient proximal tubular cells increased mitochondrial membrane potential and ammoniagenesis. In conclusion, metabolic acidosis induces autophagy in proximal tubular cells, which is indispensable for maintaining proper mitochondrial functions including ammoniagenesis, and thus for adapted urinary acid excretion. Our results provide a rationale for the beneficial effect of alkali supplementation in CKD, a condition in which autophagy may be reduced, and suggest a new therapeutic option for acidosis by modulating autophagy. Copyright © 2014 by the American Society of Nephrology.

Preparation and characterization of protein-loaded poly(epsilon-caprolactone) microparticles for oral vaccine delivery.

PubMed

Benoit, M A; Baras, B; Gillard, J

1999-07-05

This paper describes the conditions of preparation of poly(epsilon-caprolactone) (PCL) microparticles with a mean size between 5 and 10 microm, obtained by a double emulsion-solvent evaporation technique, suitable for oral vaccine delivery. Bovine serum albumin (BSA) was used as water-soluble model antigen for encapsulation. Different parameters influencing the microparticle size, the BSA loading and entrapment efficiency were investigated. Spherical, smooth and homogeneously distributed microparticles were produced with a BSA loading and entrapment efficiency reaching, respectively, 5% (w/w) and 30%. Polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) analyses of BSA released from these particles confirmed that the entrapped protein seemed to remain unaltered by the protein encapsulation process. Copyright.

Formation and functional attributes of electrostatic complexes involving casein and anionic polysaccharides: An approach to enhance oral absorption of lycopene in rats in vivo.

PubMed

Jain, Ashay; Thakur, Deepika; Ghoshal, Gargi; Katare, O P; Singh, Bhupinder; Shivhare, U S

2016-12-01

The current work entails a novel strategy of formulating the microparticles of lycopene solely using rational blends of biopolymers without using equipment-intensive techniques. The study is intended to enhance oral bioavailability of lycopene by controlling its release from micro-formulation and facilitating its absorption though lymphatic pathways. Considering the minimum particle size, maximum entrapment efficiency and loading capacity, the amounts of casein (i.e., protein) and gum tragacanth (i.e., polysaccharide) were selected as the critical factors for formulation of microparticles. Complex formation and electrostatic interaction was confirmed by Fourier transform infra red (FTIR) spectra. Size and surface properties of microparticles were studied using scanning electron microscopy (SEM). The optimized formulation (mean particle size: ∼130μm; % entrapment efficiency: ∼67% and loading capacity: ∼71%) designated noticeable improvement in lycopene release profile (over 80% in 24h). Increment in the values of C max (2.22-fold) and AUC (1.97-fold) further indicated noteworthy augmentation in the rate and extent of bioavailability by the microparticles formulation compared to plain lycopene. The resulting formulation was found to be quite stable all through two months of study episode. The resultant microparticles formulation was evaluated for antioxidant activity and tested for their effectiveness in self life enhancement of vegetable oil by calculating peroxide value under temperature and storage condition. Encapsulation strongly increased the stability of micronutrients. The current investigations, therefore, report the successful development of biopolymeric microparticles with improved bioavailability potential of lycopene. Copyright © 2016. Published by Elsevier B.V.

Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

PubMed Central

Gaur Dixit, Upasna; Barker, Jason H.; Teesch, Lynn M.; Love-Homan, Laurie; Donelson, John E.; Wilson, Mary E.

2013-01-01

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. PMID:23630964

Cy5 total protein normalization in Western blot analysis.

PubMed

Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

2015-10-01

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

Controlled release and intracellular protein delivery from mesoporous silica nanoparticles.

PubMed

Deodhar, Gauri V; Adams, Marisa L; Trewyn, Brian G

2017-01-01

Protein therapeutics are promising candidates for disease treatment due to their high specificity and minimal adverse side effects; however, targeted protein delivery to specific sites has proven challenging. Mesoporous silica nanoparticles (MSN) have demonstrated to be ideal candidates for this application, given their high loading capacity, biocompatibility, and ability to protect host molecules from degradation. These materials exhibit tunable pore sizes, shapes and volumes, and surfaces which can be easily functionalized. This serves to control the movement of molecules in and out of the pores, thus entrapping guest molecules until a specific stimulus triggers release. In this review, we will cover the benefits of using MSN as protein therapeutic carriers, demonstrating that there is great diversity in the ways MSN can be used to service proteins. Methods for controlling the physical dimensions of pores via synthetic conditions, applications of therapeutic protein loaded MSN materials in cancer therapies, delivering protein loaded MSN materials to plant cells using biolistic methods, and common stimuli-responsive functionalities will be discussed. New and exciting strategies for controlled release and manipulation of proteins are also covered in this review. While research in this area has advanced substantially, we conclude this review with future challenges to be tackled by the scientific community. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electricity forecasting on the individual household level enhanced based on activity patterns

PubMed Central

Gajowniczek, Krzysztof; Ząbkowski, Tomasz

2017-01-01

Leveraging smart metering solutions to support energy efficiency on the individual household level poses novel research challenges in monitoring usage and providing accurate load forecasting. Forecasting electricity usage is an especially important component that can provide intelligence to smart meters. In this paper, we propose an enhanced approach for load forecasting at the household level. The impacts of residents’ daily activities and appliance usages on the power consumption of the entire household are incorporated to improve the accuracy of the forecasting model. The contributions of this paper are threefold: (1) we addressed short-term electricity load forecasting for 24 hours ahead, not on the aggregate but on the individual household level, which fits into the Residential Power Load Forecasting (RPLF) methods; (2) for the forecasting, we utilized a household specific dataset of behaviors that influence power consumption, which was derived using segmentation and sequence mining algorithms; and (3) an extensive load forecasting study using different forecasting algorithms enhanced by the household activity patterns was undertaken. PMID:28423039

Electricity forecasting on the individual household level enhanced based on activity patterns.

PubMed

Gajowniczek, Krzysztof; Ząbkowski, Tomasz

2017-01-01

Leveraging smart metering solutions to support energy efficiency on the individual household level poses novel research challenges in monitoring usage and providing accurate load forecasting. Forecasting electricity usage is an especially important component that can provide intelligence to smart meters. In this paper, we propose an enhanced approach for load forecasting at the household level. The impacts of residents' daily activities and appliance usages on the power consumption of the entire household are incorporated to improve the accuracy of the forecasting model. The contributions of this paper are threefold: (1) we addressed short-term electricity load forecasting for 24 hours ahead, not on the aggregate but on the individual household level, which fits into the Residential Power Load Forecasting (RPLF) methods; (2) for the forecasting, we utilized a household specific dataset of behaviors that influence power consumption, which was derived using segmentation and sequence mining algorithms; and (3) an extensive load forecasting study using different forecasting algorithms enhanced by the household activity patterns was undertaken.

Simultaneous measurement of passage through the restriction point and MCM loading in single cells

PubMed Central

Håland, T. W.; Boye, E.; Stokke, T.; Grallert, B.; Syljuåsen, R. G.

2015-01-01

Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyper-phosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1-phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells. PMID:26250117

Protein kinase Cα (PKCα) regulates bone architecture and osteoblast activity.

PubMed

Galea, Gabriel L; Meakin, Lee B; Williams, Christopher M; Hulin-Curtis, Sarah L; Lanyon, Lance E; Poole, Alastair W; Price, Joanna S

2014-09-12

Bones' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca(-/-) female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca(-/-) but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca(-/-) mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca(-/-) mice do not. Female Prkca(-/-) mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca(-/-) mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Epigenetic dysregulation of epstein-barr virus latency and development of autoimmune disease.

PubMed

Niller, Hans Helmut; Wolf, Hans; Ay, Eva; Minarovits, Janos

2011-01-01

Epstein-Barr virus (EBV) is ahumanherpesvirus thatpersists in the memory B-cells of the majority of the world population in a latent form. Primary EBV infection is asymptomatic or causes a self-limiting disease, infectious mononucleosis. Virus latency is associated with a wide variety of neoplasms whereof some occur in immune suppressed individuals. Virus production does not occur in strict latency. The expression of latent viral oncoproteins and nontranslated RNAs is under epigenetic control via DNA methylation and histone modifications that results either in a complete silencing of the EBV genome in memory B cells, or in a cell-type dependent usage of a couple of latency promoters in tumor cells, germinal center B cells and lymphoblastoid cells (LCL, transformed by EBV in vitro). Both, latent and lytic EBV proteins elicit a strong immune response. In immune suppressed and infectious mononucleosis patients, an increased viral load can be detected in the blood. Enhanced lytic replication may result in new infection- and transformation-events and thus is a risk factor both for malignant transformation and the development of autoimmune diseases. An increased viral load or a changed presentation of a subset of lytic or latent EBV proteins that cross-react with cellular antigens may trigger pathogenic processes through molecular mimicry that result in multiple sclerosis (MS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA

PubMed Central

Wang, Ya-Yun; Tsay, Yi-Fang

2011-01-01

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport. PMID:21571952

The effects of biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with quercetin on stemness, viability and osteogenic differentiation potential of stem cell spheroids.

PubMed

Lee, H; Nguyen, T T; Kim, M; Jeong, J-H; Park, J-B

2018-05-31

Quercetin has been reported to exert many beneficial effects on the protection against various diseases, such as diabetes, cancer, and inflammation. The aim of this study is to evaluate the potential osteogenic differentiation ability of mesenchymal stem cells in the presence of quercetin. Quercetin-loaded poly(lactic-co-glycolic acid) microspheres were prepared using an electrospraying technique. Characterization of the microspheres was evaluated with a scanning electron microscope and release profile. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells. Qualitative results of cellular viability were seen under a confocal microscope, and quantitative cellular viability was evaluated using the Cell Counting Kit-8 assay. The alkaline phosphatase activity and Alizarin Red S staining were performed. A quantitative real-time polymerase chain reaction and a western blot analysis were performed. Spheroids were well formed irrespective of quercetin concentration. Most of the cells in spheroids emitted green fluorescence, and the morphology was round without significant changes. The application of quercetin-loaded microspheres produced a significant increase in the alkaline phosphatase activity. The real-time polymerase chain reaction results showed a significant increase in Runx2, and western blot results showed higher expression of Runx2 protein expression. Biodegradable microspheres loaded with quercetin produced prolonged release profiles with increased mineralization. Microspheres loaded with quercetin can be used for the enhancement of osteoblastic differentiation in cell therapy. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Visible-light-induced Ag/BiVO4 semiconductor with enhanced photocatalytic and antibacterial performance

NASA Astrophysics Data System (ADS)

Regmi, Chhabilal; Dhakal, Dipesh; Wohn Lee, Soo

2018-02-01

An Ag-loaded BiVO4 visible-light-driven photocatalyst was synthesized by the microwave hydrothermal method followed by photodeposition. The photocatalytic performance of the synthesized samples was evaluated on a mixed dye (methylene blue and rhodamine B), as well as bisphenol A in aqueous solution. Similarly, the disinfection activities of synthesized samples towards the Gram-negative Escherichia coli (E. coli) in a model cell were investigated under irradiation with visible light (λ ≥ 420 nm). The synthesized samples have monoclinic scheelite structure. Photocatalytic results showed that all Ag-loaded BiVO4 samples exhibited greater degradation and a higher mineralization rate than the pure BiVO4, probably due to the presence of surface plasmon absorption that arises due to the loading of Ag on the BiVO4 surface. The optimum Ag loading of 5 wt% has the highest photocatalytic performance and greatest stability with pseudo-first-order rate constants of 0.031 min-1 and 0.023 min-1 for the degradation of methylene blue and rhodamine B respectively in a mixture with an equal volume and concentration of each dye. The photocatalytic degradation of bisphenol A reaches 76.2% with 5 wt% Ag-doped BiVO4 within 180 min irradiation time. Similarly, the Ag-loaded BiVO4 could completely inactivate E. coli cells within 30 min under visible light irradiation. The disruption of the cell membrane as well as degradation of protein and DNA exhibited constituted evidence for antibacterial activity towards E. coli. Moreover, the bactericidal mechanisms involved in the photocatalytic disinfection process were systematically investigated.

Mechanism of action of hypoglycemic effects of an intestine-specific inhibitor of microsomal triglyceride transfer protein (MTP) in obese rats.

PubMed

Sakata, Shohei; Katsumi, Sohei; Mera, Yasuko; Kuroki, Yukiharu; Nashida, Reiko; Kakutani, Makoto; Ohta, Takeshi

2015-01-01

Diminished insulin sensitivity in the peripheral tissues and failure of pancreatic beta cells to secrete insulin are known major determinants of type 2 diabetes mellitus. JTT-130, an intestine-specific microsomal transfer protein inhibitor, has been shown to suppress high fat-induced obesity and ameliorate impaired glucose tolerance while enhancing glucagon-like peptide-1 (GLP-1) secretion. We investigated the effects of JTT-130 on glucose metabolism and elucidated the mechanism of action, direct effects on insulin sensitivity and glucose-stimulated insulin secretion in a high fat diet-induced obesity rat model. Male Sprague Dawley rats fed a high-fat diet were treated with a single administration of JTT-130. Glucose tolerance, hyperglycemic clamp and hyperinsulinemic-euglycemic testing were performed to assess effects on insulin sensitivity and glucose-stimulated insulin secretion, respectively. Plasma GLP-1 and tissue triglyceride content were also determined under the same conditions. A single administration of JTT-130 suppressed plasma glucose elevations after oral glucose loading and increased the disposition index while elevating GLP-1. JTT-130 also enhanced glucose-stimulated insulin secretion in hyperglycemic clamp tests, whereas increased insulin sensitivity was observed in hyperinsulinemic-euglycemic clamp tests. Single-dose administration of JTT-130 decreased lipid content in the liver and skeletal muscle. JTT-130 demonstrated acute and direct hypoglycemic effects by enhancing insulin secretion and/or insulin sensitivity. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Muscle Carnosine Concentration with the Co-Ingestion of Carbohydrate with β-alanine in Male Rats.

PubMed

Naderi, Alireza; Sadeghi, Mehdi; Sarshin, Amir; Imanipour, Vahid; Nazeri, Seyed Ali; Farkhayi, Fatemeh; Willems, Mark E T

2017-07-04

Muscle carnosine is an intracellular buffer. The intake of β-alanine, combined with carbohydrate and protein, enhanced carnosine loading in human muscle. The aim of the present study was to examine if muscle carnosine loading was enhanced by β-alanine intake and co-ingestion of glucose in male rats. Thirty-six male rats were divided into three groups and supplemented for four weeks: β-alanine (βA group, 1.8% β-alanine in drinking water), β-alanine and glucose (βAGL group, 1.8% β-alanine and 5% glucose in drinking water), and control (C group, drinking water). During the supplementation period, rats were exercised (20 m·min -1 , 10 min·day -1 , 4 days·week -1 for 4 weeks). Muscle carnosine concentration was quantified in soleus (n = 12) and rectus femoris (n = 6) muscles using high-performance liquid chromatography. In soleus muscle, carnosine concentration was 2.24 ± 1.10, 6.12 ± 1.08, and 6.93 ± 2.56 mmol/kg dw for control, βA, and βAGL, respectively. In rectus femoris, carnosine concentration was 2.26 ± 1.31, 7.90 ± 1.66, and 8.59 ± 2.33 mmol/kg dw for control, βA, and βAGL respectively. In each muscle, βA and βAGL resulted in similar carnosine increases compared to the control. In conclusion, β-alanine intake for four weeks, either alone or with glucose co-ingestion, equally increased muscle carnosine content. It appears that the potential insulin response to fluid glucose intake does not affect muscle carnosine loading in male rats.

Open-mouthed hybrid microcapsules with elevated enzyme loading and enhanced catalytic activity.

PubMed

Shi, Jiafu; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

2014-10-25

Open-mouthed hybrid microcapsules (HMCs) are synthesized through a hard-templating method. When utilized for enzyme immobilization and enzymatic catalysis, the open-mouthed HMCs show high enzyme loading capability, enhanced catalytic activity and desirable recycling stability, due to their fully exposed outer and inner surfaces.

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

PubMed

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

2012-08-15

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

Inactivation of viruses using novel protein A wash buffers.

PubMed

Bolton, Glen R; Selvitelli, Keith R; Iliescu, Ionela; Cecchini, Douglas J

2015-01-01

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc-fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc-fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities. © 2014 American Institute of Chemical Engineers.

Cheese whey: A potential resource to transform into bioprotein, functional/nutritional proteins and bioactive peptides.

PubMed

Yadav, Jay Shankar Singh; Yan, Song; Pilli, Sridhar; Kumar, Lalit; Tyagi, R D; Surampalli, R Y

2015-11-01

The byproduct of cheese-producing industries, cheese whey, is considered as an environmental pollutant due to its high BOD and COD concentrations. The high organic load of whey arises from the presence of residual milk nutrients. As demand for milk-derived products is increasing, it leads to increased production of whey, which poses a serious management problem. To overcome this problem, various technological approaches have been employed to convert whey into value-added products. These technological advancements have enhanced whey utilization and about 50% of the total produced whey is now transformed into value-added products such as whey powder, whey protein, whey permeate, bioethanol, biopolymers, hydrogen, methane, electricity bioprotein (single cell protein) and probiotics. Among various value-added products, the transformation of whey into proteinaceous products is attractive and demanding. The main important factor which is attractive for transformation of whey into proteinaceous products is the generally recognized as safe (GRAS) regulatory status of whey. Whey and whey permeate are biotransformed into proteinaceous feed and food-grade bioprotein/single cell protein through fermentation. On the other hand, whey can be directly processed to obtain whey protein concentrate, whey protein isolate, and individual whey proteins. Further, whey proteins are also transformed into bioactive peptides via enzymatic or fermentation processes. The proteinaceous products have applications as functional, nutritional and therapeutic commodities. Whey characteristics, and its transformation processes for proteinaceous products such as bioproteins, functional/nutritional protein and bioactive peptides are covered in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Internal Structure and Preferential Protein Binding of Colloidal Aggregates.

PubMed

Duan, Da; Torosyan, Hayarpi; Elnatan, Daniel; McLaughlin, Christopher K; Logie, Jennifer; Shoichet, Molly S; Agard, David A; Shoichet, Brian K

2017-01-20

Colloidal aggregates of small molecules are the most common artifact in early drug discovery, sequestering and inhibiting target proteins without specificity. Understanding their structure and mechanism has been crucial to developing tools to control for, and occasionally even exploit, these particles. Unfortunately, their polydispersity and transient stability have prevented exploration of certain elementary properties, such as how they pack. Dye-stabilized colloidal aggregates exhibit enhanced homogeneity and stability when compared to conventional colloidal aggregates, enabling investigation of some of these properties. By small-angle X-ray scattering and multiangle light scattering, pair distance distribution functions suggest that the dye-stabilized colloids are filled, not hollow, spheres. Stability of the coformulated colloids enabled investigation of their preference for binding DNA, peptides, or folded proteins, and their ability to purify one from the other. The coformulated colloids showed little ability to bind DNA. Correspondingly, the colloids preferentially sequestered protein from even a 1600-fold excess of peptides that are themselves the result of a digest of the same protein. This may reflect the avidity advantage that a protein has in a surface-to-surface interaction with the colloids. For the first time, colloids could be shown to have preferences of up to 90-fold for particular proteins over others. Loaded onto the colloids, bound enzyme could be spun down, resuspended, and released back into buffer, regaining most of its activity. Implications of these observations for colloid mechanisms and utility will be considered.

Barley ROP Binding Kinase1 Is Involved in Microtubule Organization and in Basal Penetration Resistance to the Barley Powdery Mildew Fungus1[W

PubMed Central

Huesmann, Christina; Reiner, Tina; Hoefle, Caroline; Preuss, Jutta; Jurca, Manuela E.; Domoki, Mónika; Fehér, Attila; Hückelhoven, Ralph

2012-01-01

Certain plant receptor-like cytoplasmic kinases were reported to interact with small monomeric G-proteins of the RHO of plant (ROP; also called RAC) family in planta and to be activated by this interaction in vitro. We identified a barley (Hordeum vulgare) partial cDNA of a ROP binding protein kinase (HvRBK1) in yeast (Saccharomyces cerevisiae) two-hybrid screenings with barley HvROP bait proteins. Protein interaction of the constitutively activated (CA) barley HvROPs CA HvRACB and CA HvRAC1 with full-length HvRBK1 was verified in yeast and in planta. Green fluorescent protein-tagged HvRBK1 appears in the cytoplasm and nucleoplasm, but CA HvRACB or CA HvRAC1 can recruit green fluorescent protein-HvRBK1 to the cell periphery. Barley HvRBK1 is an active kinase in vitro, and activity is enhanced by CA HvRACB or GTP-loaded HvRAC1. Hence, HvRBK1 might act downstream of active HvROPs. Transient-induced gene silencing of barley HvRBK1 supported penetration by the parasitic fungus Blumeria graminis f. sp. hordei, suggesting a function of the protein in basal disease resistance. Transient knockdown of HvRBK1 also influenced the stability of cortical microtubules in barley epidermal cells. Hence, HvRBK1 might function in basal resistance to powdery mildew by influencing microtubule organization. PMID:22415513

Induction of Sirt1 by Mechanical Stretch of Skeletal Muscle through the Early Response Factor EGR1 Triggers an Antioxidative Response*

PubMed Central

Pardo, Patricia S.; Mohamed, Junaith S.; Lopez, Michael A.; Boriek, Aladin M.

2011-01-01

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species (1, 2). The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription (3–5). In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging. PMID:20971845

Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens

PubMed Central

Xing, Dongxia; Li, Sufang; Robinson, Simon N.; Yang, Hong; Steiner, David; Komanduri, Krishna V.; Shpall, Elizabeth J.

2009-01-01

In the control of T-helper type I (Th-1) polarization, dendritic cells (DCs) must interpret a complex array of stimuli, many of which are poorly understood. Here we demonstrate that Th-1 polarization is heavily influenced by DC-autonomous phenomena triggered by the loading of DCs with antigenically matched major histocompatibility complex (MHC) class I and class II determinants, that is, class I and II peptide epitopes exhibiting significant amino acid sequence overlap (such as would be physiologically present during infectious processes requiring Th-1 immunity for clearance). Data were derived from 13 independent antigenic models including whole-cell systems, single-protein systems, and 3 different pairs of overlapping class I and II binding epitopes. Once loaded with matched class I and II antigens, these “Th-1 DCs” exhibited differential cytokine secretion and surface marker expression, a distinct transcriptional signature, and acquired the ability to enhance generation of CD8+ T lymphocytes. Mechanistically, tRNA-synthetases were implicated as components of a putative sensor complex involved in the comparison of class I and II epitopes. These data provide rigorous conceptual explanations for the process of Th-1 polarization and the antigenic specificity of cognate T-cell help, enhance the understanding of Th-1 responses, and should contribute to the formulation of more effective vaccination strategies. PMID:19171878

Using Gelatin Nanoparticle Mediated Intranasal Delivery of Neuropeptide Substance P to Enhance Neuro-Recovery in Hemiparkinsonian Rats.

PubMed

Zhao, Ying-Zheng; Jin, Rong-Rong; Yang, Wei; Xiang, Qi; Yu, Wen-Ze; Lin, Qian; Tian, Fu-Rong; Mao, Kai-Li; Lv, Chuan-Zhu; Wáng, Yi-Xiáng J; Lu, Cui-Tao

2016-01-01

Intranasal administration of phospholipid-based gelatin nanoparticles (GNP) was prepared to investigate the neuro-recovery effects of neuropeptide Substance P (SP) on hemiparkinsonian rats. The SP-loaded gelatin nanoparticles (SP-GNP) were prepared by a water-in-water emulsion method and possessed high stability, encapsulating efficiency and loading capacity. PC-12 cells were used to examine the growth enhancement of SP-GNP in vitro by MTT assays and flow cytometry (FCM). The therapeutic effects of SP-GNP on 6-hydroxydopamine (6-OHDA) induced hemiparkinsonian rats were assessed by quantifying rotational behavior and the levels of tyrosine hydroxylase (TH), phosphorylated c-Jun protein (p-c-Jun) and Caspase-3 (Cas-3) expressed in substantia nigra (SN) region of hemiparkinsonian rats. PC-12 cells under SP-GNP treatment showed better cell viability and lower degree of apoptosis than those under SP solution treatment. Hemiparkinsonian rats under intranasal SP-GNP administration demonstrated better behavioral improvement, higher level of TH in SN along with much lower extent of p-c-Jun and Cas-3 than those under intranasal SP solution administration and intravenous SP-GNP administration. With the advantages of GNP and nose-to-brain pathway, SP can be effectively delivered into the damaged SN region and exhibit its neuro-recovery function through the inhibition on JNK pathway and dopaminergic neuron apoptosis.

A general bio-inspired, novel interface engineering strategy toward strong yet tough protein based composites

NASA Astrophysics Data System (ADS)

Jin, Shicun; Li, Kuang; Li, Jianzhang

2018-07-01

Biopolymers show a broad prospect as an effective alternative to petroleum-based materials. However, assembling biopolymers into the composites with integrated high strength and toughness still remains a great challenge. Herein, we developed a novel and versatile mussel-inspired modification design for tough and high-performance graphene oxide (GO)/soy protein isolate (SPI) nanocomposite films, where the GO nanosheets were modified with poly(dopamine) (PDA) to improve the dispersion of GO nanosheets in SPI matrix and enhance their interfacial adhesion. As expected, at 0.6 wt% of PDA-modified GO (PDG) loading, the tensile strength and toughness of the SPI/PDG films reached 8.87 MPa and 22.82 MJ m-3, respectively, which simultaneously showed 86.34% and 263.95% higher than those of pristine film. The great enhancement of mechanical behaviors was due to the increased fracture line energy and the lack of significant coalescence of microcracks, as well as the strong interfacial adhesion force between peptide chains and PDG nanosheets. The resultant nanocomposite films also exhibited favorable vapor barrier behavior and water-resistance. The proposed method in this paper opens a new avenue for assembling two-dimensional nanosheets into the biopolymer-based composites with integrated high strength and great toughness for a series of innovative future applications.

Performance of innovative PU-foam and natural fiber-based composites for the biofiltration of a mixture of volatile organic compounds by a fungal biofilm.

PubMed

Gutiérrez-Acosta, O B; Arriaga, S; Escobar-Barrios, V A; Casas-Flores, S; Almendarez-Camarillo, A

2012-01-30

The performance of perlite and two innovative carriers that consist of polyurethane (PU) chemically modified with starch; and polypropylene reinforced with agave fibers was evaluated in the biofiltration of a mixture of VOCs composed of hexane, toluene and methyl-ethyl-ketone. At a total organic loading rate of 145 gCm(-3)h(-1) the elimination capacities (ECs) obtained were 145, 24 and 96 gCm(-3)h(-1) for the biofilters packed with the PU, the reinforced polypropylene, and perlite, respectively. Specific maximum biodegradation rates of the mixture, in the biofilters, were 416 mgCg(protein)(-1)  h(-1) for the PU and 63 mgCg(protein)(-1) h(-1) for perlite, which confirms the highest performance of the PU-composite. 18S rDNA analysis from the PU-biofilter revealed the presence of Fusarium solani in its sexual and asexual states, respectively. The modified PU carrier significantly reduced the start-up period of the biofilter and enhanced the EC of the VOCs. Thus, this study gives new alternatives in the field of packing materials synthesis, promoting the addition of easily biodegradable sources to enhance the performance of biofilters. Copyright © 2011 Elsevier B.V. All rights reserved.

Association between feline immunodeficiency virus (FIV) plasma viral RNA load, concentration of acute phase proteins and disease severity.

PubMed

Kann, Rebecca K C; Seddon, Jennifer M; Kyaw-Tanner, Myat T; Henning, Joerg; Meers, Joanne

2014-08-01

Veterinarians have few tools to predict the rate of disease progression in FIV-infected cats. In contrast, in HIV infection, plasma viral RNA load and acute phase protein concentrations are commonly used as predictors of disease progression. This study evaluated these predictors in cats naturally infected with FIV. In older cats (>5 years), log10 FIV RNA load was higher in the terminal stages of disease compared to the asymptomatic stage. There was a significant association between log10 FIV RNA load and both log10 serum amyloid A concentration and age in unwell FIV-infected cats. This study suggests that viral RNA load and serum amyloid A warrant further investigation as predictors of disease status and prognosis in FIV-infected cats. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vitro and in vivo evaluation of a water-in-oil microemulsion system for enhanced peptide intestinal delivery.

PubMed

Liu, Dongyun; Kobayashi, Taku; Russo, Steven; Li, Fengling; Plevy, Scott E; Gambling, Todd M; Carson, Johnny L; Mumper, Russell J

2013-01-01

Peptide and protein drugs have become the new generation of therapeutics, yet most of them are only available as injections, and reports on oral local intestinal delivery of peptides and proteins are quite limited. The aim of this work was to develop and evaluate a water-in-oil (w/o) microemulsion system in vitro and in vivo for local intestinal delivery of water-soluble peptides after oral administration. A fluorescent labeled peptide, 5-(and-6)-carboxytetramethylrhodamine labeled HIV transactivator protein TAT (TAMRA-TAT), was used as a model peptide. Water-in-oil microemulsions consisting of Miglyol 812, Capmul MCM, Tween 80, and water were developed and characterized in terms of appearance, viscosity, conductivity, morphology, and particle size analysis. TAMRA-TAT was loaded and its enzymatic stability was assessed in modified simulated intestinal fluid (MSIF) in vitro. In in vivo studies, TAMRA-TAT intestinal distribution was evaluated using fluorescence microscopy after TAMRA-TAT microemulsion, TAMRA-TAT solution, and placebo microemulsion were orally gavaged to mice. The half-life of TAMRA-TAT in microemulsion was enhanced nearly three-fold compared to that in the water solution when challenged by MSIF. The treatment with TAMRA-TAT microemulsion after oral administration resulted in greater fluorescence intensity in all intestine sections (duodenum, jejunum, ileum, and colon) compared to TAMRA-TAT solution or placebo microemulsion. The in vitro and in vivo studies together suggested TAMRA-TAT was better protected in the w/o microemulsion in an enzyme-containing environment, suggesting that the w/o microemulsions developed in this study may serve as a potential delivery vehicle for local intestinal delivery of peptides or proteins after oral administration.

Layer by layer assembly of a biocatalytic packaging film: lactase covalently bound to low-density polyethylene.

PubMed

Wong, Dana E; Talbert, Joey N; Goddard, Julie M

2013-06-01

Active packaging is utilized to overcome limitations of traditional processing to enhance the health, safety, economics, and shelf life of foods. Active packaging employs active components to interact with food constituents to give a desired effect. Herein we describe the development of an active package in which lactase is covalently attached to low-density polyethylene (LDPE) for in-package production of lactose-free dairy products. The specific goal of this work is to increase the total protein content loading onto LDPE using layer by layer (LbL) deposition, alternating polyethylenimine, glutaraldehyde (GL), and lactase, to enhance the overall activity of covalently attached lactase. The films were successfully oxidized via ultraviolet light, functionalized with polyethylenimine and glutaraldehyde, and layered with immobilized purified lactase. The total protein content increased with each additional layer of conjugated lactase, the 5-layer sample reaching up to 1.3 μg/cm2 . However, the increase in total protein did not lend to an increase in overall lactase activity. Calculated apparent Km indicated the affinity of immobilized lactase to substrate remains unchanged when compared to free lactase. Calculated apparent turnover numbers (kcat ) showed with each layer of attached lactase, a decrease in substrate turnover was experienced when compared to free lactase; with a decrease from 128.43 to 4.76 s(-1) for a 5-layer conjugation. Our results indicate that while LbL attachment of lactase to LDPE successfully increases total protein mass of the bulk material, the adverse impact in enzyme efficiency may limit the application of LbL immobilization chemistry for bioactive packaging use. © 2013 Institute of Food Technologists®

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, R.M.M.; Martinez, J.R.

The uptake and efflux of the isotopic tracer /sup 45/Ca were compared in dispersed submandibular acini of both control rats and rats treated with seven daily doses of reserpine (0.5 mg/kg, i.p.). Tracer uptake occurred in a time-dependent manner in both types of acini and reached 8.4 +/- 0.2 and 8.0 +/- 0.2 pmol/mg protein, respectively, in acini from control and treated animals after 60 min of incubation. Uptake of tracer was 2.35 nmol/mg DNA in control cells and 4 nmol/mg DNA in cells from treated rats at 60 min. /sup 45/Ca uptake (per mg protein) was enhanced in controlmore » acini 48% by 20 mumol/L epinephrine; 38% by 50 mumol/L carbachol; and 23% by 10 mumol/L isoproterenol. A similar order of potency was observed when uptake was expressed per mg DNA. In acini from reserpine-treated rats, /sup 45/Ca uptake (per mg protein) was increased 53% by epinephrine, 39% by isoproterenol, and only 8% by carbachol. The same enhanced effect of isoproterenol and lack of effect of carbachol were observed when uptake was calculated per mg DNA. In the absence of secretagogue, efflux of /sup 45/Ca from tracer-pre-loaded acini was larger in acini from reserpine-treated rats (53%) than in control acini (36%). Whether expressed in terms of mg protein or mg DNA, this efflux was increased in control acini 35% by epinephrine, from 25 to 28% by isoproterenol, and 17% by carbachol. In acini of reserpine-treated rats, epinephrine increased /sup 45/Ca efflux 20%, isoproterenol from 25 to 28%, and carbachol from 14 to 15%.« less

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

PubMed

Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

2004-06-01

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

Atox1 Contains Positive Residues That Mediate Membrane Association and Aid Subsequent Copper Loading

PubMed Central

Flores, Adrian G.; Unger, Vinzenz M.

2013-01-01

Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution. PMID:24036897

Atox1 contains positive residues that mediate membrane association and aid subsequent copper loading.

PubMed

Flores, Adrian G; Unger, Vinzenz M

2013-12-01

Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here, we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution.

Whey protein aerogel as blended with cellulose crystalline particles or loaded with fish oil.

PubMed

Ahmadi, Maede; Madadlou, Ashkan; Saboury, Ali Akbar

2016-04-01

Whey protein hydrogels blended with nanocrystalline and microcrystalline cellulose particles (NCC and MCC, respectively) were prepared, followed by freeze-drying, to produce aerogels. NCC blending increased the Young's modulus, and elastic character, of the protein aerogel. Aerogels were microporous and mesoporous materials, as characterized by the pores sizing 1.2 nm and 12.2 nm, respectively. Blending with NCC decreased the count of both microporous and mesoporous-classified pores at the sub-100 nm pore size range investigated. In contrast, MCC blending augmented the specific surface area and pores volume of the aerogel. It also increased moisture sorption affinity of aerogel. The feasibility of conveying hydrophobic nutraceuticals by aerogels was evaluated through loading fish oil into the non-blended aerogel. Oil loading altered its microstructure, corresponding to a peak displacement in Fourier-transform infra-red spectra, which was ascribed to increased hydrophobic interactions. Surface coating of aerogel with zein decreased the oxidation susceptibility of the loaded oil during subsequent storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dissociable Roles of Different Types of Working Memory Load in Visual Detection

PubMed Central

Konstantinou, Nikos; Lavie, Nilli

2013-01-01

We contrasted the effects of different types of working memory (WM) load on detection. Considering the sensory-recruitment hypothesis of visual short-term memory (VSTM) within load theory (e.g., Lavie, 2010) led us to predict that VSTM load would reduce visual-representation capacity, thus leading to reduced detection sensitivity during maintenance, whereas load on WM cognitive control processes would reduce priority-based control, thus leading to enhanced detection sensitivity for a low-priority stimulus. During the retention interval of a WM task, participants performed a visual-search task while also asked to detect a masked stimulus in the periphery. Loading WM cognitive control processes (with the demand to maintain a random digit order [vs. fixed in conditions of low load]) led to enhanced detection sensitivity. In contrast, loading VSTM (with the demand to maintain the color and positions of six squares [vs. one in conditions of low load]) reduced detection sensitivity, an effect comparable with that found for manipulating perceptual load in the search task. The results confirmed our predictions and established a new functional dissociation between the roles of different types of WM load in the fundamental visual perception process of detection. PMID:23713796

Wide-field in vivo neocortical calcium dye imaging using a convection-enhanced loading technique combined with simultaneous multiwavelength imaging of voltage-sensitive dyes and hemodynamic signals

PubMed Central

Ma, Hongtao; Harris, Samuel; Rahmani, Redi; Lacefield, Clay O.; Zhao, Mingrui; Daniel, Andy G. S.; Zhou, Zhiping; Bruno, Randy M.; Berwick, Jason; Schwartz, Theodore H.

2014-01-01

Abstract. In vivo calcium imaging is an incredibly powerful technique that provides simultaneous information on fast neuronal events, such as action potentials and subthreshold synaptic activity, as well as slower events that occur in the glia and surrounding neuropil. Bulk-loading methods that involve multiple injections can be used for single-cell as well as wide-field imaging studies. However, multiple injections result in inhomogeneous loading as well as multiple sites of potential cortical injury. We used convection-enhanced delivery to create smooth, continuous loading of a large area of the cortical surface through a solitary injection site and demonstrated the efficacy of the technique using confocal microscopy imaging of single cells and physiological responses to single-trial events of spontaneous activity, somatosensory-evoked potentials, and epileptiform events. Combinations of calcium imaging with voltage-sensitive dye and intrinsic signal imaging demonstrate the utility of this technique in neurovascular coupling investigations. Convection-enhanced loading of calcium dyes may be a useful technique to advance the study of cortical processing when widespread loading of a wide-field imaging is required. PMID:25525611

Wide-field in vivo neocortical calcium dye imaging using a convection-enhanced loading technique combined with simultaneous multiwavelength imaging of voltage-sensitive dyes and hemodynamic signals.

PubMed

Ma, Hongtao; Harris, Samuel; Rahmani, Redi; Lacefield, Clay O; Zhao, Mingrui; Daniel, Andy G S; Zhou, Zhiping; Bruno, Randy M; Berwick, Jason; Schwartz, Theodore H

2014-07-24

In vivo calcium imaging is an incredibly powerful technique that provides simultaneous information on fast neuronal events, such as action potentials and subthreshold synaptic activity, as well as slower events that occur in the glia and surrounding neuropil. Bulk-loading methods that involve multiple injections can be used for single-cell as well as wide-field imaging studies. However, multiple injections result in inhomogeneous loading as well as multiple sites of potential cortical injury. We used convection-enhanced delivery to create smooth, continuous loading of a large area of the cortical surface through a solitary injection site and demonstrated the efficacy of the technique using confocal microscopy imaging of single cells and physiological responses to single-trial events of spontaneous activity, somatosensory-evoked potentials, and epileptiform events. Combinations of calcium imaging with voltage-sensitive dye and intrinsic signal imaging demonstrate the utility of this technique in neurovascular coupling investigations. Convection-enhanced loading of calcium dyes may be a useful technique to advance the study of cortical processing when widespread loading of a wide-field imaging is required.

Negative viscosity can enhance learning of inertial dynamics.

PubMed

Huang, Felix C; Patton, James L; Mussa-Ivaldi, Ferdinando A

2009-06-01

We investigated how learning of inertial load manipulation is influenced by movement amplification with negative viscosity. Using a force-feedback device, subjects trained on anisotropic loads (5 orientations) with free movements in one of three conditions (inertia only, negative viscosity only, or combined), prior to common evaluation conditions (prescribed circular pattern with inertia only). Training with Combined-Load resulted in lower error (6.89±3.25%) compared to Inertia-Only (8.40±4.32%) and Viscosity-Only (8.17±4.13%) according to radial deviation analysis (% of trial mean radius). Combined-Load and Inertia-Only groups exhibited similar unexpected no-load trials (8.38±4.31% versus 8.91±4.70% of trial mean radius), which suggests comparable low-impedance strategies. These findings are remarkable since negative viscosity, only available during training, evidently enhanced learning when combined with inertia. Modeling analysis suggests that a feedforward after-effect of negative viscosity cannot predict such performance gains. Instead, results from Combined-Load training are consistent with greater feedforward inertia compensation along with a small increase in impedance control. The capability of the nervous system to generalize learning from negative viscosity suggests an intriguing new method for enhancing sensorimotor adaptation.

Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

PubMed

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C; Brown, Andrew J; Sandoval, Cecilia; Hallab, Jeannette C; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-03-14

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

PubMed Central

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-01-01

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

Nanocarrier-mediated foliar zinc fertilization influences expression of metal homeostasis related genes in flag leaves and enhances gluten content in durum wheat.

PubMed

Deshpande, Paresh; Dapkekar, Ashwin; Oak, Manoj; Paknikar, Kishore; Rajwade, Jyutika

2018-01-01

Wheat is the staple food for most of the world's population; however, it is a poor source of zinc. Foliar fertilization of zinc via zinc loaded chitosan nanocarriers (Zn-CNP) post-anthesis has proved to be a promising approach for grain zinc enhancement in durum wheat as evidenced in our earlier study. However, the molecular mechanism of uptake of zinc via Zn-CNP remains unclear. Foliar application of Zn-CNP was performed at post anthesis stages in two durum wheat cultivars (MACS 3125 and UC1114, containing the Gpc-B1 gene), and expression levels of several metal-related genes were analyzed during early senescence. Zn-CNP application indeed caused changes in gene expression as revealed by qPCR data on representative genes involved in metal homeostasis, phloem transporters, and leaf senescence. Furthermore, zinc-regulated transporters and iron (Fe)-regulated transporter-like protein (ZIP) family [ZIP1, ZIP7, ZIP15], CA (carbonic anhydrase), and DMAS (2'-deoxymugineic acid synthase) in flag leaves exhibited significant correlation with zinc content in the seeds. The analysis of grain endosperm proteins showed enhancement of gamma gliadins while other gluten subunits decreased. Gene expression within ZIP family members varied with the type of cultivar mostly attributed to the Gpc-B1, concentration of external zinc ions as well as the type of tissue analyzed. Correlation analysis revealed the involvement of the selected genes in zinc enhancement. At the molecular level, uptake of zinc via Zn-CNP nanocarrier was comparable to the uptake of zinc via common zinc fertilizers i.e. ZnSO4.

Stimulation of the follicular bulge LGR5+ and LGR6+ stem cells with the gut-derived human alpha defensin 5 results in decreased bacterial presence, enhanced wound healing, and hair growth from tissues devoid of adnexal structures.

PubMed

Lough, Denver; Dai, Hui; Yang, Mei; Reichensperger, Joel; Cox, Lisa; Harrison, Carrie; Neumeister, Michael W

2013-11-01

Discovery of leucine-rich repeat-containing G-protein-coupled receptors 5 and 6 (LGR5 and LGR6) as markers of adult epithelial stem cells of the skin and intestine permits researchers to draw on the intrinsic cellular fundamentals of wound healing and proliferation dynamics of epithelial surfaces. In this study, the authors use the intestine-derived human alpha defensin 5 to stimulate epithelial proliferation, bacterial reduction, and hair production in burn wound beds to provide the field with initial insight on augmenting wound healing in tissues devoid of adnexal stem cells. Murine third-degree burn wound beds were treated with (1) intestine-derived human alpha defensin 5, (2) skin-derived human beta defensin 1, and (3) sulfadiazine to determine their roles in wound healing, bacterial reduction, and hair growth. The human alpha defensin 5 peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin 1 and sulfadiazine. Human alpha defensin 5 was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed significant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wisp1. Ex vivo studies showed enhanced cell migration in human alpha defensin 5-treated wounds compared with controls. Application of human alpha defensin 5 increases LGR stem cell migration into wound beds, leading to enhanced healing, bacterial reduction, and hair production through the augmentation of key Wnt and wound healing transcripts. These findings can be used to derive gut protein-based therapeutics in wound healing.

In vivo distribution and antitumor activity of heparin-stabilized doxorubicin-loaded liposomes.

PubMed

Han, Hee Dong; Lee, Aeri; Song, Chung Kil; Hwang, Taewon; Seong, Hasoo; Lee, Chong Ock; Shin, Byung Cheol

2006-04-26

The purpose of this study was to investigate the effect of heparin conjugation to the surface of doxorubicin (DOX)-loaded liposomes on the circulation time, biodistribution and antitumor activity after intravenous injection in murine B16F10 melanoma tumor-bearing mice. The heparin-conjugated liposomes (heparin-liposomes) were prepared by fixation of the negatively charged heparin to the positively charged liposomes. The existence of heparin on the liposomal surface was confirmed by measuring the changes in the particle size, zeta potential and heparin amount of the liposomes. The stability of the heparin-liposomes in serum was higher than that of the control liposomes, due to the heparin-liposomes being better protected from the adsorption of serum proteins. The DOX-loaded heparin-liposomes showed high drug levels for up to 64 h after the intravenous injection and the half-life of DOX was approximately 8.4- or 1.5-fold higher than that of the control liposomes or polyethyleneglycol-fixed liposomes (PEG-liposomes), respectively. The heparin-liposomes accumulated to a greater extent in the tumor than the control or PEG-liposomes as a result of their lower uptake by the reticuloendothelial system cells in the liver and spleen. In addition, the DOX-loaded heparin-liposomes retarded the growth of the tumor effectively compared with the control or PEG-liposomes. These results indicate the promising potential of heparin-liposomes as a new sterically stabilized liposomal delivery system for the enhancement of the therapeutic efficacy of chemotherapeutic agents.

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

USGS Publications Warehouse

Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

2011-01-01

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

NASA Astrophysics Data System (ADS)

Borisova, T. A.; Himmelreich, N. H.

The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

[Modeling of experimental hypertension by chronic salt loading combined with a low-protein diet in Wistar rats].

PubMed

Strekalova, V V; Khachirov, D G; Dedenkov, A N; Suvorov, Iu I; Shvatsabaia, I K

1989-01-01

Combination of chronic salt loading with protein-poor diet produces experimental hypertension with natrium consumption near to physiological. The present model is characterized, compared to the existing one, by stage development, moderate arterial blood pressure elevation and absence of "salt toxicosis" and may be thus considered more adequate for experimental investigation of primary arterial hypertension pathophysiology.

Very Low-Protein Diet (VLPD) Reduces Metabolic Acidosis in Subjects with Chronic Kidney Disease: The "Nutritional Light Signal" of the Renal Acid Load.

PubMed

Di Iorio, Biagio Raffaele; Di Micco, Lucia; Marzocco, Stefania; De Simone, Emanuele; De Blasio, Antonietta; Sirico, Maria Luisa; Nardone, Luca

2017-01-17

Metabolic acidosis is a common complication of chronic kidney disease; current guidelines recommend treatment with alkali if bicarbonate levels are lower than 22 mMol/L. In fact, recent studies have shown that an early administration of alkali reduces progression of CKD. The aim of the study is to evaluate the effect of fruit and vegetables to reduce the acid load in CKD. We conducted a case-control study in 146 patients who received sodium bicarbonate. Of these, 54 patients assumed very low-protein diet (VLPD) and 92 were controls (ratio 1:2). We calculated every three months the potential renal acid load (PRAL) and the net endogenous acid production (NEAP), inversely correlated with serum bicarbonate levels and representing the non-volatile acid load derived from nutrition. Un-paired T -test and Chi-square test were used to assess differences between study groups at baseline and study completion. Two-tailed probability values ≤0.05 were considered statistically significant. At baseline, there were no statistical differences between the two groups regarding systolic blood pressure (SBP), diastolic blood pressure (DBP), protein and phosphate intake, urinary sodium, potassium, phosphate and urea nitrogen, NEAP, and PRAL. VLPD patients showed at 6 and 12 months a significant reduction of SBP ( p < 0.0001), DBP ( p < 0.001), plasma urea ( p < 0.0001) protein intake ( p < 0.0001), calcemia ( p < 0.0001), phosphatemia ( p < 0.0001), phosphate intake ( p < 0.0001), urinary sodium ( p < 0.0001), urinary potassium ( p < 0.002), and urinary phosphate ( p < 0.0001). NEAP and PRAL were significantly reduced in VLPD during follow-up. VLPD reduces intake of acids; nutritional therapy of CKD, that has always taken into consideration a lower protein, salt, and phosphate intake, should be adopted to correct metabolic acidosis, an important target in the treatment of CKD patients. We provide useful indications regarding acid load of food and drinks-the "acid load dietary traffic light".

Reversible polyelectrolyte capsules as carriers for protein delivery.

PubMed

Anandhakumar, S; Nagaraja, V; Raichur, Ashok M

2010-07-01

A reversible drug delivery system based on spontaneous deposition of a model protein into preformed microcapsules has been demonstrated for protein delivery applications. Layer-by-Layer assembly of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) onto polystyrene sulfonate (PSS) doped CaCO3 particles, followed by core removal yielded intact hollow microcapsules having a unique property to induce spontaneous deposition of bovine serum albumin (BSA) at pH below its isoelectric point of 4.8, where it was positively charged. These capsules showed reversible pH dependent open and closed states to fluorescence labeled dextran (FITC-Dextran) and BSA (FITC-BSA). The loading capacity of BSA increased from 9.1 x 10(7) to 2.03 x 10(8) molecules per capsule with decrease in pH from 4.5 to 3. The loading of BSA-FITC was observed by confocal laser scanning microscopy (CLSM), which showed homogeneous distribution of protein inside the capsule. Efficient loading of BSA was further confirmed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The interior capsule concentration was as high as 209 times the feeding concentration when the feeding concentration was increased from 1 to 10 mg/ml. The deposition was initially controlled by spontaneous loading mechanism at lower BSA concentration followed by diffusion controlled loading at higher concentration; which decreased the loading efficiency from 35% to 7%. Circular dichroism (CD) measurements and Fourier transform infrared spectroscopy (FTIR) confirmed that there was no significant change in conformation of released BSA in comparison with native BSA. The release was initially burst in the first 0.5 h and sustained up to 5 h. The hollow capsules were found to be biocompatible with mouse embryonic fibroblast (MEF) cells during in vitro cell culture studies. Thus these pH sensitive polyelectrolyte microcapsules may offer a promising delivery system for water soluble proteins and peptides. 2010 Elsevier B.V. All rights reserved.

Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation

PubMed Central

Xu, Wen-Hong; Han, Min; Dong, Qi; Fu, Zhi-Xuan; Diao, Yuan-Yuan; Liu, Hai; Xu, Jing; Jiang, Hong-Liang; Zhang, Su-Zhan; Zheng, Shu; Gao, Jian-Qing; Wei, Qi-Chun

2012-01-01

Background The purpose of this study is to evaluate the efficacy of composite doxorubicinloaded micelles for enhancing doxorubicin radiosensitivity in multicellular spheroids from a non-small cell lung cancer cell line. Methods A novel composite doxorubicin-loaded micelle consisting of polyethylene glycolpolycaprolactone/Pluronic P105 was developed, and carrier-mediated doxorubicin accumulation and release from multicellular spheroids was evaluated. We used confocal laser scanning microscopy and flow cytometry to study the accumulation and efflux of doxorubicin from A549 multicellular spheroids. Doxorubicin radiosensitization and the combined effects of irradiation and doxorubicin on cell migration and proliferation were compared for the different doxorubicin delivery systems. Results Confocal laser scanning microscopy and quantitative flow cytometry studies both verified that, for equivalent doxorubicin concentrations, composite doxorubicin-loaded micelles significantly enhanced cellular doxorubicin accumulation and inhibited doxorubicin release. Colony-forming assays demonstrated that composite doxorubicin-loaded micelles are radiosensitive, as shown by significantly reduced survival of cells treated by radiation + composite micelles compared with those treated with radiation + free doxorubicin or radiation alone. The multicellular spheroid migration area and growth ability verified higher radiosensitivity for the composite micelles loaded with doxorubicin than for free doxorubicin. Conclusion Our composite doxorubicin-loaded micelle was demonstrated to have radiosensitization. Doxorubicin loading in the composite micelles significantly increased its cellular uptake, improved drug retention, and enhanced its antitumor effect relative to free doxorubicin, thereby providing a novel approach for treatment of cancer. PMID:22679376

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Bradley R.; Drake, Eric J.; Shi, Ce

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived frommore » understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.« less

Partially oxidized polyvinyl alcohol as a promising material for tissue engineering.

PubMed

Stocco, Elena; Barbon, Silvia; Grandi, Francesca; Gamba, Pier Giorgio; Borgio, Luca; Del Gaudio, Costantino; Dalzoppo, Daniele; Lora, Silvano; Rajendran, Senthilkumar; Porzionato, Andrea; Macchi, Veronica; Rambaldo, Anna; De Caro, Raffaele; Parnigotto, Pier Paolo; Grandi, Claudio

2017-07-01

The desired clinical outcome after implantation of engineered tissue substitutes depends strictly on the development of biodegradable scaffolds. In this study we fabricated 1% and 2% oxidized polyvinyl alcohol (PVA) hydrogels, which were considered for the first time for tissue-engineering applications. The final aim was to promote the protein release capacity and biodegradation rate of the resulting scaffolds in comparison with neat PVA. After physical crosslinking, characterization of specific properties of 1% and 2% oxidized PVA was performed. We demonstrated that mechanical properties, hydrodynamic radius of molecules, thermal characteristics and degree of crystallinity were inversely proportional to the PVA oxidation rate. On the other hand, swelling behaviour and protein release were enhanced, confirming the potential of oxidized PVA as a protein delivery system, besides being highly biodegradable. Twelve weeks after in vivo implantation in mice, the modified hydrogels did not elicit severe inflammatory reactions, showing them to be biocompatible and to degrade faster as the degree of oxidation increased. According to our results, oxidized PVA stands out as a novel biomaterial for tissue engineering that can be used to realize scaffolds with customizable mechanical behaviour, protein-loading ability and biodegradability. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Electrostatic assembly of binary nanoparticle superlattices using protein cages

NASA Astrophysics Data System (ADS)

Kostiainen, Mauri A.; Hiekkataipale, Panu; Laiho, Ari; Lemieux, Vincent; Seitsonen, Jani; Ruokolainen, Janne; Ceci, Pierpaolo

2013-01-01

Binary nanoparticle superlattices are periodic nanostructures with lattice constants much shorter than the wavelength of light and could be used to prepare multifunctional metamaterials. Such superlattices are typically made from synthetic nanoparticles, and although biohybrid structures have been developed, incorporating biological building blocks into binary nanoparticle superlattices remains challenging. Protein-based nanocages provide a complex yet monodisperse and geometrically well-defined hollow cage that can be used to encapsulate different materials. Such protein cages have been used to program the self-assembly of encapsulated materials to form free-standing crystals and superlattices at interfaces or in solution. Here, we show that electrostatically patchy protein cages--cowpea chlorotic mottle virus and ferritin cages--can be used to direct the self-assembly of three-dimensional binary superlattices. The negatively charged cages can encapsulate RNA or superparamagnetic iron oxide nanoparticles, and the superlattices are formed through tunable electrostatic interactions with positively charged gold nanoparticles. Gold nanoparticles and viruses form an AB8fcc crystal structure that is not isostructural with any known atomic or molecular crystal structure and has previously been observed only with large colloidal polymer particles. Gold nanoparticles and empty or nanoparticle-loaded ferritin cages form an interpenetrating simple cubic AB structure (isostructural with CsCl). We also show that these magnetic assemblies provide contrast enhancement in magnetic resonance imaging.

Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

PubMed

Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

2007-06-01

Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture*

PubMed Central

Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M.

2016-01-01

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. PMID:27597544

Acid-degradable lactobionic acid-modified soy protein nanogels crosslinked by ortho ester linkage for efficient antitumor in vivo.

PubMed

Cheng, Xu; Qin, Jiejie; Wang, Xin; Zha, Qian; Yao, Weijing; Fu, Shengxiang; Tang, Rupei

2018-05-03

It remains a crucial challenge to achieve efficient cellular uptake and intracellular drug release in tumor cells for the nanoscale drug delivery systems. Herein, acid-degradable nanogels were prepared by cross-linking methacrylated soy protein with an acid-labile ortho ester cross-linker (NG1), and then modified with lactobionic acid (LA) to give tumor-targeted nanogels (NG2). Both NG1 and NG2 displayed excellent stability in neutral environment, while showed pH-triggered degradation behaviors under mildly acidic conditions resulting from the breakage of ortho ester bonds. Doxorubicin (DOX) was successfully loaded into nanogels, which exhibited an accelerated release at low pH. In vitro cell studies demonstrated that LA-modified nanogels could effectively improve cellular internalization, show higher cytotoxicity and apoptosis toward asialoglycoprotein receptor (ASGPR) over-expressed HepG2 cells. In vivo antitumor experimentproved that LA modification could significantly enhance the tumor-targeting ability of nanogels and increase DOX concentration in tumor site, leading to better therapeutic efficacy. Histological analysis further demonstrated that soy protein-based nanogels did not cause any damage to normal organs. Overall, these pH-sensitive and tumor-targeting soy protein-based nanogels can be potential drug carriers for efficient tumor treatment. Copyright © 2018. Published by Elsevier B.V.

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture.

PubMed

Miller, Bradley R; Drake, Eric J; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2016-10-21

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of household cooking techniques on the microbiological load and the nutritional quality of mealworms (Tenebrio molitor L. 1758).

PubMed

Caparros Megido, Rudy; Poelaert, Christine; Ernens, Marjorie; Liotta, Marisa; Blecker, Christophe; Danthine, Sabine; Tyteca, Eva; Haubruge, Éric; Alabi, Taofic; Bindelle, Jérôme; Francis, Frédéric

2018-04-01

Mealworms are new food products in Europe, but consumers do not know how to cook them. Although cooking could increase the safety, acceptability, palatability, and digestibility of insects, the heating process could have deleterious effects on protein and lipid quality. Therefore, this study characterized the effects of different household cooking methods (boiling, pan-frying, vacuum cooking, and oven cooking) on the microbial load and nutritive value of mealworms, with a focus on protein digestibility and fatty acid composition. Boiling and cooking under vacuum were the most efficient techniques to reduce microbial load while maintaining the high levels of protein and polyunsaturated fatty acids of mealworms. Cooking method-related changes were very low on macronutrients content except for pan-fried mealworms which exhibited the highest lipid content. Cooking slightly changed fatty acid composition of mealworms by principally decreasing their level of saturated fatty acids but also increased the in vitro crude protein digestibility of mealworms. Copyright © 2018 Elsevier Ltd. All rights reserved.

Leucine/Pd-loaded (5,5) single-walled carbon nanotube matrix as a novel nanobiosensors for in silico detection of protein.

PubMed

Yoosefian, Mehdi; Etminan, Nazanin

2018-06-01

We have designed a novel nanobiosensor for in silico detecting proteins based on leucine/Pd-loaded single-walled carbon nanotube matrix. Density functional theory at the B3LYP/6-31G (d) level of theory was realized to analyze the geometrical and electronic structure of the proposed nanobiosensor. The solvent effects were investigated using the Tomasi's polarized continuum model. Atoms-in-molecules theory was used to study the nature of interactions by calculating the electron density ρ(r) and Laplacian at the bond critical points. Natural bond orbital analysis was performed to achieve a deep understanding of the nature of the interactions. The biosensor has potential application for high sensitive and rapid response to protein due to the chemical adsorption of L-leucine amino acid onto Pd-loaded single-walled carbon nanotube and reactive functional groups that can incorporate in hydrogen binding, hydrophobic interactions and van der Waals forces with the protein surface in detection process.

Block Copolymer Micelles as Nanocontainers for Controlled Release of Proteins from Biocompatible Oil Phases

PubMed Central

2009-01-01

Biocompatible oils are used in a variety of medical applications ranging from vaccine adjuvants to vehicles for oral drug delivery. To enable such nonpolar organic phases to serve as reservoirs for delivery of hydrophilic compounds, we explored the ability of block copolymer micelles in organic solvents to sequester proteins for sustained release across an oil−water interface. Self-assembly of the block copolymer, poly(ϵ-caprolactone)-block-poly(2-vinyl pyridine) (PCL-b-P2VP), was investigated in toluene and oleic acid, a biocompatible naturally occurring fatty acid. Micelle formation in toluene was characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM) imaging of micelles cast onto silicon substrates. Cryogenic transmission electron microscopy confirmed a spherical morphology in oleic acid. Studies of homopolymer solubility implied that micelles in oleic acid consist of a P2VP corona and a PCL core, while P2VP formed the core of micelles assembled in toluene. The loading of two model proteins (ovalbumin (ova) and bovine serum albumin (BSA)) into micelles was demonstrated with loadings as high as 7.8% wt of protein per wt of P2VP in oleic acid. Characterization of block copolymer morphology in the two solvents after protein loading revealed spherical particles with similar size distributions to the as-assembled micelles. Release of ova from micelles in oleic acid was sustained for 12−30 h upon placing the oil phase in contact with an aqueous bath. Unique to the situation of micelle assembly in an oily phase, the data suggest protein is sequestered in the P2VP corona block of PCL-b-P2VP micelles in oleic acid. More conventionally, protein loading occurs in the P2VP core of micelles assembled in toluene. PMID:19235932

Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design.

PubMed

Lagoutte, Priscillia; Mignon, Charlotte; Stadthagen, Gustavo; Potisopon, Supanee; Donnat, Stéphanie; Mast, Jan; Lugari, Adrien; Werle, Bettina

2018-05-11

In the past decades protein nanoparticles have successfully been used for vaccine applications. Their particulate nature and dense repetitive subunit organization makes them perfect carriers for antigen surface display and confers high immunogenicity. Nanoparticles have emerged as excellent candidates for vectorization of biological and immunostimulating molecules. Nanoparticles and biomolecular nanostructures such as ferritins or virus like particles have been used as diagnostic and therapeutic delivery systems, in vaccine development, as nanoreactors, etc. Recently, a new class of bacterial protein compartment has been discovered referred to as encapsulin nanocompartment. These compartments have been used for targeted diagnostics, as therapeutic delivery systems and as nanoreactors. Their biological origin makes them conveniently biocompatible and allows genetic functionalization. The aim of our study was to implement encapsulin nanocompartements for simultaneous epitope surface display and heterologous protein loading for rational vaccine design. For this proof-of-concept-study, we produced Thermotoga maritima encapsulin nanoparticles in E. coli. We demonstrated the ability of simultaneous display in our system by inserting Matrix protein 2 ectodomain (M2e) of influenza A virus at the nanoparticle surface and by packaging of a fluorescent reporter protein (GFP) into the internal cavity. Characterization of the nanoparticles by electronic microscopy confirmed homogenously shaped particles of 24 nm diameter in average. The results further show that engineering of the particle surface improved the loading capacity of the heterologous reporter protein suggesting that surface display may induce a critical elastic deformation resulting in improved stiffness. In Balb/c mice, nanoparticle immunization elicited antibody responses against both the surface epitope and the loaded cargo protein. These results confirm the potential of encapsulin nanocompartments for customized vaccine design and antigen delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Piezoelectric control of columns prone to instabilities and nonlinear modal interaction

NASA Astrophysics Data System (ADS)

Sridharan, Srinivasan; Kim, Sunjung

2008-06-01

This paper attempts to unravel the issues of piezoelectric control of structures prone to nonlinear static and dynamic instabilities. A simple yet typical example is considered, namely the problem of a simply supported axially compressed imperfect column on an elastic softening foundation. Here the significant nonlinearity arises from the softening foundation. The column is so designed as to have coincident critical loads for the first two modes of buckling. Piezoelectric actuators/sensors are deemed to be attached to a column in regions of maximum strain at several locations along the length of the column. The issues involved in (i) enhancing the static buckling load, (ii) suppression of vibrations as the column is compressed to a load close to its dynamic instability load and (iii) enhancing the dynamic instability load are investigated and discussed. It is shown that there is a premium price to pay for enhancing the buckling capacity of the column, be it static or dynamic. The paper concludes by alluding to the possibility of a failure of patch control if a higher-order shortwave mode happens to be the governing principal mode of the structure.

Enhancement of urban heat load through social inequalities on an example of a fictional city King's Landing.

PubMed

Žuvela-Aloise, M

2017-03-01

The numerical model MUKLIMO_3 is used to simulate the urban climate of an imaginary city as an illustrative example to demonstrate that the residential areas with deprived socio-economic conditions can exhibit an enhanced heat load at night, and thus more disadvantageous environmental conditions, compared with the areas of higher socio-economic status. The urban climate modelling simulations differentiate between orographic, natural landscape, building and social effects, where social differences are introduced by selection of location, building type and amount of vegetation. The model results show that the increase of heat load can be found in the areas inhabited by the poor population as a combined effect of natural and anthropogenic factors. The unfavourable location in the city and the building type, consisting of high density, low housing with high fraction of pavement and small amount of vegetation contribute to the formation of excessive heat load. This abstract example shows that the enhancement of urban heat load can be linked to the concept of a socially stratified city and is independent of the historical development of any specific city.

Enhancement of urban heat load through social inequalities on an example of a fictional city King's Landing

NASA Astrophysics Data System (ADS)

Žuvela-Aloise, M.

2017-03-01

The numerical model MUKLIMO_3 is used to simulate the urban climate of an imaginary city as an illustrative example to demonstrate that the residential areas with deprived socio-economic conditions can exhibit an enhanced heat load at night, and thus more disadvantageous environmental conditions, compared with the areas of higher socio-economic status. The urban climate modelling simulations differentiate between orographic, natural landscape, building and social effects, where social differences are introduced by selection of location, building type and amount of vegetation. The model results show that the increase of heat load can be found in the areas inhabited by the poor population as a combined effect of natural and anthropogenic factors. The unfavourable location in the city and the building type, consisting of high density, low housing with high fraction of pavement and small amount of vegetation contribute to the formation of excessive heat load. This abstract example shows that the enhancement of urban heat load can be linked to the concept of a socially stratified city and is independent of the historical development of any specific city.

Efficacy, Safety and Anticancer Activity of Protein Nanoparticle-Based Delivery of Doxorubicin through Intravenous Administration in Rats

PubMed Central

Golla, Kishore; Cherukuvada, Bhaskar; Ahmed, Farhan; Kondapi, Anand K.

2012-01-01

Background and Aims Doxorubicin is a potent anticancer drug and a major limiting factor that hinders therapeutic use as its high levels of systemic circulation often associated with various off-target effects, particularly cardiotoxicity. The present study focuses on evaluation of the efficacy of doxorubicin when it is loaded into the protein nanoparticles and delivered intravenously in rats bearing Hepatocellular carcinoma (HCC). The proteins selected as carrier were Apotransferrin and Lactoferrin, since the receptors for these two proteins are known to be over expressed on cancer cells due to their iron transport capacity. Methods Doxorubicin loaded apotransferrin (Apodoxonano) and lactoferrin nanoparticles (Lactodoxonano) were prepared by sol-oil chemistry. HCC in the rats was induced by 100 mg/l of diethylnitrosamine (DENA) in drinking water for 8 weeks. Rats received 5 doses of 2 mg/kg drug equivalent nanoparticles through intravenous administration. Pharmacokinetics and toxicity of nanoformulations was evaluated in healthy rats and anticancer activity was studied in DENA treated rats. The anticancer activity was evaluated through counting of the liver nodules, H & E analysis and by estimating the expression levels of angiogenic and antitumor markers. Results In rats treated with nanoformulations, the numbers of liver nodules were found to be significantly reduced. They showed highest drug accumulation in liver (22.4 and 19.5 µg/g). Both nanoformulations showed higher localization compared to doxorubicin (Doxo) when delivered in the absence of a carrier. Higher amounts of Doxo (195 µg/g) were removed through kidney, while Apodoxonano and Lactodoxonano showed only a minimal amount of removal (<40 µg/g), suggesting the extended bioavailability of Doxo when delivered through nanoformulation. Safety analysis shows minimal cardiotoxicity due to lower drug accumulation in heart in the case of nanoformulation. Conclusion Drug delivery through nanoformulations not only minimizes the cardiotoxicity of doxorubicin but also enhances the efficacy and bioavailability of the drug in a target-specific manner. PMID:23284832

pH and Glucose Dual-Responsive Injectable Hydrogels with Insulin and Fibroblasts as Bioactive Dressings for Diabetic Wound Healing.

PubMed

Zhao, Lingling; Niu, Lijing; Liang, Hongze; Tan, Hui; Liu, Chaozong; Zhu, Feiyan

2017-11-01

pH and glucose dual-responsive injectable hydrogels were prepared through the cross-linking of Schiff's base and phenylboronate ester using phenylboronic-modified chitosan, poly(vinyl alcohol) and benzaldehyde-capped poly(ethylene glycol). Protein drugs and live cells could be incorporated into the hydrogels during the in situ cross-linking, displaying sustained and pH/glucose-triggered drug release from the hydrogels and cell viability and proliferation in the three-dimensional hydrogel matrix as well. Hence, the hydrogels with insulin and fibroblasts were considered as bioactive dressings for diabetic wound healing. A streptozotocin-induced diabetic rat model was used to evaluate the efficacy of hydrogel dressings in wound repair. The results revealed that the incorporation of insulin and L929 in the hydrogels could promote neovascularization and collagen deposition and enhance the wound-healing process of diabetic wounds. Thus, the drug- and cell-loaded hydrogels have promising potential in wound healing as a medicated system for various therapeutic proteins and live cells.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka

2016-08-29

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-familymore » replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.« less

Diet-induced acidosis and alkali supplementation.

PubMed

Della Guardia, Lucio; Roggi, Carla; Cena, Hellas

2016-11-01

Western diet, high in protein-rich foods and poor in vegetables, is likely to be responsible for the development of a moderate acid excess leading to metabolism deregulation and the onset or worsening of chronic disturbances. Available findings seem to suggest that diets with high protein/vegetables ratio are likely to induce the development of calcium lithiasis, especially in predisposed subjects. Moreover, some evidence supports the hypothesis of bone metabolism worsening and enhanced bone loss following acid-genic diet consumption although available literature seems to lack direct and conclusive evidence demonstrating pathological bone loss. According to other evidences, diet-induced acidosis is likely to induce or accelerate muscle wasting or sarcopenia, especially among elderlies. Furthermore, recent epidemiological findings highlight a specific role of dietary acid load in glucose metabolism deregulation and insulin resistance. The aim of this review is to investigate the role of acid-genic diets in the development of the mentioned metabolic disorders focusing on the possible clinical improvements exerted by alkali supplementation.

High dietary protein intake and protein-related acid load on bone health

USDA-ARS?s Scientific Manuscript database

Protein is an essential nutrient for humans and is required for maintaining optimal bone structure and growth. Consumption of high protein diets in excess of the Recommended Dietary Allowance of (0.8 g protein/kg body weight/d) is increasingly popular due to the benefits of protein on preserving lea...

Deep Tissue Injury in Development of Pressure Ulcers: A Decrease of Inflammasome Activation and Changes in Human Skin Morphology in Response to Aging and Mechanical Load

PubMed Central

Stojadinovic, Olivera; Minkiewicz, Julia; Sawaya, Andrew; Bourne, Jonathan W.; Torzilli, Peter; de Rivero Vaccari, Juan Pablo; Dietrich, W. Dalton; Keane, Robert W.; Tomic-Canic, Marjana

2013-01-01

Molecular mechanisms leading to pressure ulcer development are scarce in spite of high mortality of patients. Development of pressure ulcers that is initially observed as deep tissue injury is multifactorial. We postulate that biomechanical forces and inflammasome activation, together with ischemia and aging, may play a role in pressure ulcer development. To test this we used a newly-developed bio-mechanical model in which ischemic young and aged human skin was subjected to a constant physiological compressive stress (load) of 300 kPa (determined by pressure plate analyses of a person in a reclining position) for 0.5–4 hours. Collagen orientation was assessed using polarized light, whereas inflammasome proteins were quantified by immunoblotting. Loaded skin showed marked changes in morphology and NLRP3 inflammasome protein expression. Sub-epidermal separations and altered orientation of collagen fibers were observed in aged skin at earlier time points. Aged skin showed significant decreases in the levels of NLRP3 inflammasome proteins. Loading did not alter NLRP3 inflammasome proteins expression in aged skin, whereas it significantly increased their levels in young skin. We conclude that aging contributes to rapid morphological changes and decrease in inflammasome proteins in response to tissue damage, suggesting that a decline in the innate inflammatory response in elderly skin could contribute to pressure ulcer pathogenesis. Observed morphological changes suggest that tissue damage upon loading may not be entirely preventable. Furthermore, newly developed model described here may be very useful in understanding the mechanisms of deep tissue injury that may lead towards development of pressure ulcers. PMID:23967056

Sclerostin's role in bone's adaptive response to mechanical loading.

PubMed

Galea, Gabriel L; Lanyon, Lance E; Price, Joanna S

2017-03-01

Mechanical loading is the primary functional determinant of bone mass and architecture, and osteocytes play a key role in translating mechanical signals into (re)modelling responses. Although the precise mechanisms remain unclear, Wnt signalling pathway components, and the anti-osteogenic canonical Wnt inhibitor Sost/sclerostin in particular, play an important role in regulating bone's adaptive response to loading. Increases in loading-engendered strains down-regulate osteocyte sclerostin expression, whereas reduced strains, as in disuse, are associated with increased sclerostin production and bone loss. However, while sclerostin up-regulation appears to be necessary for the loss of bone with disuse, the role of sclerostin in the osteogenic response to loading is more complex. While mice unable to down-regulate sclerostin do not gain bone with loading, Sost knockout mice have an enhanced osteogenic response to loading. The molecular mechanisms by which osteocytes sense and transduce loading-related stimuli into changes in sclerostin expression remain unclear but include several, potentially interlinked, signalling cascades involving periostin/integrin, prostaglandin, estrogen receptor, calcium/NO and Igf signalling. Deciphering the mechanisms by which changes in the mechanical environment regulate sclerostin production may lead to the development of therapeutic strategies that can reverse the skeletal structural deterioration characteristic of disuse and age-related osteoporosis and enhance bones' functional adaptation to loading. By enhancing the osteogenic potential of the context in which individual therapies such as sclerostin antibodies act it may become possible to both prevent and reverse the age-related skeletal structural deterioration characteristic of osteoporosis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Dietary Protein and Potassium, Diet-Dependent Net Acid Load, and Risk of Incident Kidney Stones.

PubMed

Ferraro, Pietro Manuel; Mandel, Ernest I; Curhan, Gary C; Gambaro, Giovanni; Taylor, Eric N

2016-10-07

Protein and potassium intake and the resulting diet-dependent net acid load may affect kidney stone formation. It is not known whether protein type or net acid load is associated with risk of kidney stones. We prospectively examined intakes of protein (dairy, nondairy animal, and vegetable), potassium, and animal protein-to-potassium ratio (an estimate of net acid load) and risk of incident kidney stones in the Health Professionals Follow-Up Study ( n =42,919), the Nurses' Health Study I ( n =60,128), and the Nurses' Health Study II ( n =90,629). Multivariable models were adjusted for age, body mass index, diet, and other factors. We also analyzed cross-sectional associations with 24-hour urine ( n =6129). During 3,108,264 person-years of follow-up, there were 6308 incident kidney stones. Dairy protein was associated with lower risk in the Nurses' Health Study II (hazard ratio for highest versus lowest quintile, 0.84; 95% confidence interval, 0.73 to 0.96; P value for trend <0.01). The hazard ratios for nondairy animal protein were 1.15 (95% confidence interval, 0.97 to 1.36; P value for trend =0.04) in the Health Professionals Follow-Up Study and 1.20 (95% confidence interval, 0.99 to 1.46; P value for trend =0.06) in the Nurses' Health Study I. Potassium intake was associated with lower risk in all three cohorts (hazard ratios from 0.44 [95% confidence interval, 0.36 to 0.53] to 0.67 [95% confidence interval, 0.57 to 0.78]; P values for trend <0.001). Animal protein-to-potassium ratio was associated with higher risk ( P value for trend =0.004), even after adjustment for animal protein and potassium. Higher dietary potassium was associated with higher urine citrate, pH, and volume ( P values for trend <0.002). Kidney stone risk may vary by protein type. Diets high in potassium or with a relative abundance of potassium compared with animal protein could represent a means of stone prevention. Copyright © 2016 by the American Society of Nephrology.

Dietary Protein and Potassium, Diet–Dependent Net Acid Load, and Risk of Incident Kidney Stones

PubMed Central

Mandel, Ernest I.; Curhan, Gary C.; Gambaro, Giovanni; Taylor, Eric N.

2016-01-01

Background and objectives Protein and potassium intake and the resulting diet–dependent net acid load may affect kidney stone formation. It is not known whether protein type or net acid load is associated with risk of kidney stones. Design, setting, participants, & measurements We prospectively examined intakes of protein (dairy, nondairy animal, and vegetable), potassium, and animal protein-to-potassium ratio (an estimate of net acid load) and risk of incident kidney stones in the Health Professionals Follow-Up Study (n=42,919), the Nurses’ Health Study I (n=60,128), and the Nurses’ Health Study II (n=90,629). Multivariable models were adjusted for age, body mass index, diet, and other factors. We also analyzed cross-sectional associations with 24-hour urine (n=6129). Results During 3,108,264 person-years of follow-up, there were 6308 incident kidney stones. Dairy protein was associated with lower risk in the Nurses’ Health Study II (hazard ratio for highest versus lowest quintile, 0.84; 95% confidence interval, 0.73 to 0.96; P value for trend <0.01). The hazard ratios for nondairy animal protein were 1.15 (95% confidence interval, 0.97 to 1.36; P value for trend =0.04) in the Health Professionals Follow-Up Study and 1.20 (95% confidence interval, 0.99 to 1.46; P value for trend =0.06) in the Nurses’ Health Study I. Potassium intake was associated with lower risk in all three cohorts (hazard ratios from 0.44 [95% confidence interval, 0.36 to 0.53] to 0.67 [95% confidence interval, 0.57 to 0.78]; P values for trend <0.001). Animal protein-to-potassium ratio was associated with higher risk (P value for trend =0.004), even after adjustment for animal protein and potassium. Higher dietary potassium was associated with higher urine citrate, pH, and volume (P values for trend <0.002). Conclusions Kidney stone risk may vary by protein type. Diets high in potassium or with a relative abundance of potassium compared with animal protein could represent a means of stone prevention. PMID:27445166

Shock enhancement of cellular materials subjected to intensive pulse loading

NASA Astrophysics Data System (ADS)

Zhang, J.; Fan, J.; Wang, Z.; Zhao, L.; Li, Z.

2018-03-01

Cellular materials can dissipate a large amount of energy due to their considerable stress plateau, which contributes to their extensive applications in structural design for crashworthiness. However, in some experiments with specimens subjected to intense impact loads, transmitted stress enhancement has been observed, leading to severe damage to the objects protected. Transmitted stress through two-dimensional Voronoi cellular materials as a protective device is qualitatively studied in this paper. Dimensionless parameters of material properties and loading parameters are defined to give critical conditions for shock enhancement and clarify the correlation between the deformations and stress enhancement. The effect of relative density on this amplifying phenomenon is investigated as well. In addition, local strain fields are calculated by using the optimal local deformation gradient, which gives a clear presentation of deformations and possible local non-uniformity in the crushing process. This research provides valuable insight into the reliability of cellular materials as protective structures.

Strongly Modulated Friction of a Film-Terminated Ridge-Channel Structure.

PubMed

He, Zhenping; Hui, Chung-Yuen; Levrard, Benjamin; Bai, Ying; Jagota, Anand

2016-05-26

Natural contacting surfaces have remarkable surface mechanical properties, which has led to the development of bioinspired surface structures using rubbery materials with strongly enhanced adhesion and static friction. However, sliding friction of structured rubbery surfaces is almost always significantly lower than that of a flat control, often due to significant loss of contact. Here we show that a film-terminated ridge-channel structure can strongly enhance sliding friction. We show that with properly chosen materials and geometrical parameters the near surface structure undergoes mechanical instabilities along with complex folding and sliding of internal interfaces, which is responsible for the enhancement of sliding friction. Because this structure shows no enhancement of adhesion under normal indentation by a sphere, it breaks the connection between energy loss during normal and shear loading. This makes it potentially interesting in many applications, for instance in tires, where one wishes to minimize rolling resistance (normal loading) while maximizing sliding friction (shear loading).

Engineering analysis of lightweight high-opacity newsprint production by fiber loading

Treesearch

John H. Klungness; Matthew L. Stroika; Marguerite S. Sykes; Said M. Abubakr; Werner Witek; Oliver U. Heise

1999-01-01

We estimated the capital effectiveness of fiber loading in regard to producing lightweight high-opacity newsprint. Fiber loading enhances fiber bonding at increased precipitated calcium carbonate levels without significant loss in Canadian standard freeness or additional use of energy. We investigated the return on investment (ROI) for fiber loading precipitated...

High working memory load impairs the effect of cognitive reappraisal on emotional response: Evidence from an event-related potential study.

PubMed

Gan, Shuzhen; Yang, Jianfeng; Chen, Xuhai; Zhang, Xiuping; Yang, Yufang

2017-02-03

This study investigates how the working memory (WM) load influenced the efficacy of cognitive reappraisal, a frequently used strategy for emotion regulation. In a dual-task paradigm, the participants were required to perform a high-load or a low-load memory task and simultaneously reappraise aversive pictures with a negative or a neutral meaning. In the low-load condition, we found that the amplitude of emotion-enhanced late positive potential (LPP) was significantly decreased by neutral reappraisal compared to negative reappraisal. In the high-load condition, this regulatory effect of reappraisal disappeared. These results suggest that successful reappraisal relies on cognitive resources and WM processes. If the necessary resources involved in reappraisal are over-depleted by a concurrent memory task, the reappraisal effect will be impaired. Moreover, we found that emotion-enhanced LPP was significant in both of the high-load and low-load tasks, which suggests that emotional electrocortical response may not be susceptible to the available resources. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Plasmon mediated cathodic photocurrent generation in sol-gel synthesized doped SrTiO{sub 3} nanofilms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugavaneshwar, Ramu Pasupathi, E-mail: r.p.sugavaneshwar@nims.go.jp, E-mail: NAGAO.Tadaaki@nims.go.jp; Chen, Kai; Lakshminarayana, Gandham

2015-11-01

Thin films of SrTiO{sub 3} (STO) and Rh-doped SrTiO{sub 3} (Rh-STO) were synthesized by sol-gel method and loaded with Ag nanoparticles. Pristine STO films exhibited anodic photocurrent while Rh-STO exhibited cathodic photocurrent. An enhancement in the overall cathodic photocurrent is observed with Ag nanoparticle loading and an additional enhancement in the visible light range is seen from the incident photon-to-current efficiency spectrum due to synergetic effect of Rh doping and Ag loading in STO.

Ferritin Decorated PLGA/Paclitaxel Loaded Nanoparticles Endowed with an Enhanced Toxicity Toward MCF-7 Breast Tumor Cells.

PubMed

Turino, Ludmila N; Ruggiero, Maria R; Stefanìa, Rachele; Cutrin, Juan C; Aime, Silvio; Geninatti Crich, Simonetta

2017-04-19

Polylactic and glycolic acid nanoparticles (PLGA-NPs), coated with L-ferritin, are exploited for the simultaneous delivery of paclitaxel and an amphiphilic Gd based MRI contrast agent into breast cancer cells (MCF7). L-ferritin has been covalently conjugated to the external surface of PLGA-NPs exploiting NHS activated carboxylic groups. The results confirmed that nanoparticles decorated with L-ferritin have many advantages with respect to both albumin-decorated and nondecorated particles. Ferritin moieties endow PLGA-NPs with targeting capability, exploiting SCARA5 receptors overexpressed by these tumor cells, that results in an increased paclitaxel cytotoxicity. Moreover, protein coating increased nanoparticle stability, thus reducing the fast and aspecific drug release before reaching the target. The theranostic potential of the nanoparticles has been demonstrated by evaluating the signal intensity enhancement on T 1 -weighted MRI images of labeled MCF7 cells. The results were compared with that obtained with MDA cells used as negative control due to their lower SCARA5 expression.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

PubMed

Wang, Xiuli; Cui, Fuai; Madhu, Vedavathi; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2011-02-01

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

BMP-7 Preserves Surface Integrity of Degradable-ceramic Cranioplasty in a Göttingen Minipig Model

PubMed Central

Schulz, Peter; Klünter, Tim; Deisinger, Ulrike; Diez, Claudius; Waiss, Waltraud; Kirschneck, Christian; Reichert, Torsten E.; Detsch, Rainer

2017-01-01

Background: The aim of the study was to evaluate the integrity of a craniotomy grafted site in a minipig model using different highly porous calcium phosphate ceramic scaffolds either loaded or nonloaded with bone morphogenetic protein-7 (BMP-7). Methods: Four craniotomies with a diameter of 15 mm (critical-size defect) were grafted with different highly porous (92–94 vol%) calcium phosphate ceramics [hydroxyapatite (HA), tricalcium phosphate (TCP), and biphasic calcium phosphate (BCP; a mixture of HA and TCP)] in 10 Göttingen minipigs: (a) group I (n = 5): HA versus BCP; (b) group II (n = 5): TCP versus BCP. One scaffold of each composition was supplied with 250 μg of BMP-7. In vivo computed tomography scan and fluorochrome bone labeling were performed. Specimens were evaluated 14 weeks after surgery by environmental scanning electron microscopy, fluorescence microscopy, and Giemsa staining histology. Results: BMP-7 significantly enhanced bone formation in TCP (P = 0.047). Slightly enhanced bone formation was observed in BCP (P = 0.059) but not in HA implants. BMP-7 enhanced ceramic degradation in TCP (P = 0.05) and BCP (P = 0.05) implants but not in HA implants. Surface integrity of grafted site was observed in all BMP-7-loaded implants after successful creeping substitution by the newly formed bone. In 9 of 10 HA implants without BMP-7, partial collapse of the implant site was observed. All TCP implants without BMP-7 collapsed. Fluorescent labeling showed bone formation at week 1 in BMP-7-stimulated implants. Conclusions: BMP-7 supports bone formation, ceramic degradation, implant integration, and surface integrity of the grafted site. PMID:28458969

Multifunctional polyamidoamine-modified selenium nanoparticles dual-delivering siRNA and cisplatin to A549/DDP cells for reversal multidrug resistance.

PubMed

Zheng, Wenjing; Cao, Chengwen; Liu, Yanan; Yu, Qianqian; Zheng, Chuping; Sun, Dongdong; Ren, Xiaofan; Liu, Jie

2015-01-01

Multidrug resistance (MDR) is a major barrier against effective cancer treatment. Dual-delivering a therapeutic small interfering RNA (siRNA) and chemotherapeutic agents has been developed to reverse drug resistance in tumor cells. In this study, amine-terminated generation 5 polyamidoamine (PAMAM) dendrimers (G5.NH2)-modified selenium nanoparticles (G5@Se NP) were synthesized for the systemic dual-delivery of mdr1 siRNA and cisplatin (cis-diamminedichloroplatinum-(II), DDP), which was demonstrated to enhance siRNA loading, releasing efficiency and gene-silencing efficacy. When the mdr1 siRNA was conjugated with G5@Se NP via electrostatic interaction, a significant down-regulation of P-glycoprotein and multidrug resistance-associated protein expression was observed; G5@Se-DDP-siRNA arrested A549/DDP cells at G1 phase and led to enhanced cytotoxicity in A549/DDP cells through induction of apoptosis involving the AKT and ERK signaling pathways. Interestingly, G5@Se-DDP NP were much less reactive than DDP in the reactions with both MT and GSH, indicating that loading of DDP in a nano-delivery system could effectively prevent cell detoxification. Furthermore, animal studies demonstrated that the new delivery system of G5@Se-DDP-siRNA significantly enhanced the anti-tumor effect on tumor-bearing nude mice, with no appreciable abnormality in the major organs. These results suggest that G5@Se NP could be a potential platform to combine chemotherapy and gene therapy technology in the treatment of human disease. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Glycyrrhetinic acid-decorated and reduction-sensitive micelles to enhance the bioavailability and anti-hepatocellular carcinoma efficacy of tanshinone IIA.

PubMed

Chen, Fengqian; Zhang, Jinming; He, Yao; Fang, Xiefan; Wang, Yitao; Chen, Meiwan

2016-01-01

It remains a challenge to increase drug tumor-specific accumulation as well as to achieve intracellular-controlled drug release for hepatocellular carcinoma (HCC) chemotherapy. Herein, we developed a dual-functional biodegradable micellar system constituted by glycyrrhetinic acid coupling poly(ethylene glycol)-disulfide linkage-poly(lactic-co-glycolic acid) (GA-PEG-SS-PLGA) to achieve both hepatoma-targeting and redox-responsive intracellular drug release. Tanshinone IIA (TAN IIA), an effective anti-HCC drug, was encapsulated. Notably, it exhibited rapid aggregation and faster drug release in 10 mM dithiothreitol compared with the redox-insensitive control. Furthermore, GA-decorated micelles revealed HCC-specific cellular uptake in human liver cancer HepG2 cells with an energy-dependent manner, in which micropinocytosis and caveolae-mediated endocytosis were demonstrated as the major cellular pathways. The enhanced cytotoxicity and pro-apoptotic effects against HepG2 cells in vitro were observed, mediated by up-regulation of the intracellular ROS level, the increased cell cycle arrest at S phase, enhanced necrocytosis and up-regulation of caspase 3/7, P38 protein expression. In addition, TAN IIA-loaded micelles had a significantly prolonged circulation time, improved bioavailability, and resulted in an increased accumulation of TAN IIA in the liver. With the synergistic effects of HCC-targeting and controlled drug release, TAN IIA-loaded GA-PEG-SS-PLGA micelles significantly inhibited tumor growth and increased survival time in a mouse HCC-xenograft model. Collectively, the GA-PEG-SS-PLGA micelles with HCC-targeting and redox-sensitive characters would provide a novel strategy to deliver TAN IIA effectively for HCC therapy.

Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

PubMed

Hancock, Meaghan H; Cliffe, Anna R; Knipe, David M; Smiley, James R

2010-02-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

Herpes Simplex Virus VP16, but Not ICP0, Is Required To Reduce Histone Occupancy and Enhance Histone Acetylation on Viral Genomes in U2OS Osteosarcoma Cells▿ †

PubMed Central

Hancock, Meaghan H.; Cliffe, Anna R.; Knipe, David M.; Smiley, James R.

2010-01-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure. PMID:19939931

Standardization of formulations for the acute amino acid depletion and loading test

PubMed Central

Badawy, Abdulla A-B; Dougherty, Donald M

2017-01-01

The acute tryptophan (Trp) depletion (ATD) and loading (ATL) and the acute tyrosine (Tyr) plus phenylalanine (Phe) depletion (ATPD) tests are powerful tools for studying the roles of cerebral monoamines in behaviour and symptoms related to various disorders. The tests use either amino acid mixtures or proteins. Current amino acid mixtures lack specificity in humans, but not in rodents, because of the faster disposal of branched-chain amino acids (BCAA) by the latter. The high content of BCAA (30-60%) is responsible for the poor specificity in humans and we recommend, in a 50g dose, the control formulation of Young et al. (1985) with a lowered BCAA content (18%) and minor modifications as a common control for the above tests. With protein-based formulations, α-lactalbumin is specific for ATL, whereas gelatine is only partially effective for ATD. We recommend the use of the whey protein fraction glycomacropeptide (GMP) as an alternative protein. Its BCAA content is ideal for specificity and the absence of Trp, Tyr and Phe render it suitable as a template for 7 formulations (separate and combined depletion or loading and a truly balanced control). We invite the research community to participate in standardization of the depletion and loading methodologies by using our recommended amino acid formulation and developing those based on GMP. PMID:25586395

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL).

PubMed

Kwon, Young Min; Chung, Hee Sun; Moon, Cheol; Yockman, James; Park, Yoon Jeong; Gitlin, Scott D; David, Allan E; Yang, Victor C

2009-11-03

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Protein removal from waste brines generated during ham salting through acidification and centrifugation.

PubMed

Gutiérrez-Martínez, Maria del Rosario; Muñoz-Guerrero, Hernán; Alcaína-Miranda, Maria Isabel; Barat, José Manuel

2014-03-01

The salting step in food processes implies the production of large quantities of waste brines, having high organic load, high conductivity, and other pollutants with high oxygen demand. Direct disposal of the residual brine implies salinization of soil and eutrophication of water. Since most of the organic load of the waste brines comes from proteins leaked from the salted product, precipitation of dissolved proteins by acidification and removal by centrifugation is an operation to be used in waste brine cleaning. The aim of this study is optimizing the conditions for carrying out the separation of proteins from waste brines generated in the pork ham salting operation, by studying the influence of pH, centrifugal force, and centrifugation time. Models for determining the removal of proteins depending on the pH, centrifugal force, and time were obtained. The results showed a high efficacy of the proposed treatment for removing proteins, suggesting that this method could be used for waste brine protein removal. The best pH value to be used in an industrial process seems to be 3, while the obtained results indicate that almost 90% of the proteins from the brine can be removed by acidification followed by centrifugation. A further protein removal from the brine should have to be achieved using filtrating techniques, which efficiency could be highly improved as a consequence of the previous treatment through acidification and centrifugation. Waste brines from meat salting have high organic load and electrical conductivity. Proteins can be removed from the waste brine by acidification and centrifugation. The total protein removal can be up to 90% of the initial content of the waste brine. Protein removal is highly dependent on pH, centrifugation rate, and time. © 2014 Institute of Food Technologists®

pH-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: Effects of protein size, crosslinking density, and hydrogel degradation on protein delivery.

PubMed

Koetting, Michael Clinton; Guido, Joseph Frank; Gupta, Malvika; Zhang, Annie; Peppas, Nicholas A

2016-01-10

Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. pH-responsive poly(itaconic acid-co-N-vinyl-2-pyrrolidone) (P(IA-co-NVP)) hydrogel microparticles were tested in vitro with model proteins salmon calcitonin, urokinase, and rituximab to determine the effects of particle size, protein size, and crosslinking density on oral delivery capability. Particle size showed no significant effect on overall delivery potential but did improve percent release of encapsulated protein over the micro-scale particle size range studied. Protein size was shown to have a significant impact on the delivery capability of the P(IA-co-NVP) hydrogel. We show that when using P(IA-co-NVP) hydrogel microparticles with 3 mol% tetra(ethylene glycol) dimethacrylate crosslinker, a small polypeptide (salmon calcitonin) loads and releases up to 45 μg/mg hydrogel while the mid-sized protein urokinase and large monoclonal antibody rituximab load and release only 19 and 24 μg/mg hydrogel, respectively. We further demonstrate that crosslinking density offers a simple method for tuning hydrogel properties to variously sized proteins. Using 5 mol% TEGDMA crosslinker offers optimal performance for the small peptide, salmon calcitonin, whereas lower crosslinking density of 1 mol% offers optimal performance for the much larger protein rituximab. Finally, an enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK were synthesized and tested in simulated gastric and intestinal conditions. These hydrogels offer ideal loading and release behavior, showing no degradative release of encapsulated salmon calcitonin in gastric conditions while yielding rapid and complete release of encapsulated protein within 1h in intestinal conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

New doxorubicin nanocarriers based on cyclodextrins.

PubMed

Viale, Maurizio; Giglio, Valentina; Monticone, Massimiliano; Maric, Irena; Lentini, Giovanni; Rocco, Mattia; Vecchio, Graziella

2017-10-01

Polymeric nanoparticles and fibrin gels (FBGs) are attractive biomaterials for local delivery of a variety of biotherapeutic agents, from drugs to proteins. We combined these different drug delivery approaches by preparing nanoparticle-loaded FBGs characterized by their intrinsic features of drug delivery rate and antiproliferative/apoptotic activities. Inclusion complexes of doxorubicin (DOXO) with oligomeric β-cyclodextrins (oCyD) functionalized with different functional groups were studied. These nanocarriers were able to interact with FBGs as shown by a decreased release rate of DOXO. One of these complexes, oCyDNH 2 /DOXO, demonstrated good antiproliferative and apoptotic activity in vitro, reflecting a higher drug uptake by cells. As hypothesized, the nanocarrier/FBG complexes showed a lower drug release rate than similar FBGs loaded with the corresponding non-functionalized oCyD/DOXO. Taken together, our results provide experimental evidence that oCyDNH 2 /DOXO complexes may be useful components in enhanced FBGs and further build support for the great promise these complex molecules hold for clinical use in localized anticancer therapy of inoperable or surgically removable tumors of different histological origin.

Self-assembled polymeric nanoparticles as new, smart contrast agents for cancer early detection using magnetic resonance imaging.

PubMed

Mouffouk, Fouzi; Simão, Teresa; Dornelles, Daniel F; Lopes, André D; Sau, Pablo; Martins, Jorge; Abu-Salah, Khalid M; Alrokayan, Salman A; Rosa da Costa, Ana M; dos Santos, Nuno R

2015-01-01

Early cancer detection is a major factor in the reduction of mortality and cancer management cost. Here we developed a smart and targeted micelle-based contrast agent for magnetic resonance imaging (MRI), able to turn on its imaging capability in the presence of acidic cancer tissues. This smart contrast agent consists of pH-sensitive polymeric micelles formed by self-assembly of a diblock copolymer (poly(ethyleneglycol-b-trimethylsilyl methacrylate)), loaded with a gadolinium hydrophobic complex ((t)BuBipyGd) and exploits the acidic pH in cancer tissues. In vitro MRI experiments showed that (t)BuBipyGd-loaded micelles were pH-sensitive, as they turned on their imaging capability only in an acidic microenvironment. The micelle-targeting ability toward cancer cells was enhanced by conjugation with an antibody against the MUC1 protein. The ability of our antibody-decorated micelles to be switched on in acidic microenvironments and to target cancer cells expressing specific antigens, together with its high Gd(III) content and its small size (35-40 nm) reveals their potential use for early cancer detection by MRI.

Preparation and investigation of Ulex europaeus agglutinin I-conjugated liposomes as potential oral vaccine carriers.

PubMed

Li, KeXin; Chen, DaWei; Zhao, XiuLi; Hu, HaiYang; Yang, ChunRong; Pang, DaHai

2011-11-01

We prepared and optimized Ulex europaeus agglutinin I (UEAI)-modified Bovine serum albumin (BSA)-encapsulating liposomes (UEAI-LIP) as oral vaccine carriers and examined the feasibility of inducing systemic and mucosal immune responses by oral administration of UEAILIP. The prepared systems were characterized in vitro for their average size, zeta potential, encapsulation efficiency (EE%) and conjugation efficiency (CE%). In vitro release studies indicated that the presence of UEAI around the optimized liposomes was able to prevent a burst release of loaded BSA and provide sustained release of the encapsulated protein. In vivo immune-stimulating results in KM mice showed that BSA given intramuscularly generated systemic response only but both systemic and mucosal immune responses could be induced simultaneously in the groups in which BSA-loaded liposomes (LIP) and UEAI-LIP were administered intragastrically. Furthermore, the modification of UEAI on the surface of liposomes could further enhance the IgA and IgG levels obviously. In conclusion, this study demonstrated the high potential of lectin-modified liposomes containing the antigen as carriers for oral vaccine.

Performance nutrition guidelines for international rugby sevens tournaments.

PubMed

Dziedzic, Christine E; Higham, Dean G

2014-06-01

Rugby sevens is an abbreviated version of rugby union, played by teams of seven players over 7-min halves. International competitions are usually played in a tournament format. While shorter in duration, the movement demands of rugby sevens per min of match time are greater than rugby union, resulting in an accentuated load on players. This load can be repeated up to six times over a typical 2- or 3-day competition period. The potential cumulative effect of inadequate carbohydrate, protein and/or fluid intake over the course of a tournament is the greatest nutrition-related concern for players. Nutritional strategies before and during competition are suggested to replenish substrate stores, maintain fluid balance and promote recovery between matches. The use of ergogenic aids known to enhance intermittent, high-intensity activity and/or the execution of motor skills may be advantageous to rugby sevens performance and is discussed. This review provides a best-practice model of nutritional support for international rugby sevens competition based on our current understanding of the sport combined with pragmatic guidelines and considerations for the practitioner.

Nuclear and cytoplasmic delivery of lactoferrin in glioma using chitosan nanoparticles: Cellular location dependent-action of lactoferrin.

PubMed

Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

2018-08-01

Lactoferrin (Lf) exerts anti-cancer effects on glioma, however, the exact mechanism remains unclear. Despite possessing a nuclear localization sequence (NLS), Lf was found to allocate only in the cytoplasm of glioma 261. Lf was therefore loaded into nuclear and cytoplasmic targeted nanoparticles (NPs) to determine whether nuclear delivery of Lf would enhance its anti-cancer effect. Upon treatment with 300 and 800 µg/mL Lf loaded chitosan NPs, nuclear targeted Lf-NPs showed 1.3 and 2.7 folds increase in cell viability, whereas cytoplasmic targeted Lf-NPs at 300 µg/mL decreased cell viability by 0.8 folds in comparison to free Lf and controls. Results suggest that the cytotoxicity of Lf on glioma is attributable to its cytoplasmic allocation. Nuclear delivery of Lf induced cell proliferation rather than cytotoxicity, indicating that the mode of action of Lf in glioma is cell location dependent. This calls for caution about the general use of Lf as an anti-cancer protein. Copyright © 2018. Published by Elsevier B.V.

The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis.

PubMed

Chandra, Richard P; Chu, QiuLu; Hu, Jinguang; Zhong, Na; Lin, Mandy; Lee, Jin-Suk; Saddler, Jack

2016-01-01

With the goal of enhancing overall carbohydrate recovery and reducing enzyme loading refiner mechanical pulping and steam pretreatment (210°C, 5 min) were used to pretreat poplar wood chips. Neutral sulphonation post-treatment indicated that, although the lignin present in the steam pretreated substrate was less reactive, the cellulose-rich, water insoluble component was more accessible to cellulases and Simons stain. This was likely due to lignin relocation as the relative surface lignin measured by X-ray photoelectron spectroscopy increased from 0.4 to 0.8. The integration of sulphite directly into steam pretreatment resulted in the solubilisation of 60% of the lignin while more than 80% of the carbohydrate present in the original substrate was recovered in the water insoluble fraction after Na2CO3 addition. More than 80% of the sugars present in the original cellulose and xylan could be recovered after 48 h using an enzyme loading of 20 mg protein/g cellulose at a 10% substrate concentration. Copyright © 2015 Elsevier Ltd. All rights reserved.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

PubMed

Zhang, Lu; Mo, Jinyao; Swanson, Karen V; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom; Shu, Hong-Bing; Duncan, Joseph A; Ting, Jenny P-Y

2014-03-20

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP. Copyright © 2014 Elsevier Inc. All rights reserved.

Use of Capillaries for Macromolecular Crystallization in a Cryogenic Dewar

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Hammons, Aaron S.; Hong, Young Soo

2002-01-01

The enhanced gaseous nitrogen (EGN) dewar is a cryogenic dry shipper with a sealed cylinder inserted inside along with a temperature monitoring device, and is intended for macromolecular crystallization experiments on the International Space Station. Within the dewar, each crystallization experiment is contained as a solution within a plastic capillary tube. The standard procedure for loading samples in these tubes has involved rapid freezing of the precipitant and biomolecular solution, e.g., protein, directly in liquid nitrogen; this method, however, often resulted in uncontrolled formation of air voids, These air pockets, or bubbles, can lead to irreproducible crystallization results. A novel protocol has been developed to prevent formation of bubbles, and this has been tested in the laboratory as well as aboard the International Space Station during a 42-day long mission of July/August 2001. The gain or loss of mass from solutions within the plastic capillaries revealed that mass transport occurred among separated tubes, and that this mass transport was dependent upon the hygroscopic character of the solution contained in any given tube. The surface area of the plastic capillary tube also related to the observed mass transport. Furthermore, the decreased mass of solutions of-protein correlated to observed formation of protein crystals.

A crossmodal crossover: opposite effects of visual and auditory perceptual load on steady-state evoked potentials to irrelevant visual stimuli.

PubMed

Jacoby, Oscar; Hall, Sarah E; Mattingley, Jason B

2012-07-16

Mechanisms of attention are required to prioritise goal-relevant sensory events under conditions of stimulus competition. According to the perceptual load model of attention, the extent to which task-irrelevant inputs are processed is determined by the relative demands of discriminating the target: the more perceptually demanding the target task, the less unattended stimuli will be processed. Although much evidence supports the perceptual load model for competing stimuli within a single sensory modality, the effects of perceptual load in one modality on distractor processing in another is less clear. Here we used steady-state evoked potentials (SSEPs) to measure neural responses to irrelevant visual checkerboard stimuli while participants performed either a visual or auditory task that varied in perceptual load. Consistent with perceptual load theory, increasing visual task load suppressed SSEPs to the ignored visual checkerboards. In contrast, increasing auditory task load enhanced SSEPs to the ignored visual checkerboards. This enhanced neural response to irrelevant visual stimuli under auditory load suggests that exhausting capacity within one modality selectively compromises inhibitory processes required for filtering stimuli in another. Copyright © 2012 Elsevier Inc. All rights reserved.

ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders

PubMed Central

Nam, Hyeyoung; Wang, Chia-Yu; Zhang, Lin; Zhang, Wei; Hojyo, Shintaro; Fukada, Toshiyuki; Knutson, Mitchell D.

2013-01-01

The liver, pancreas, and heart are particularly susceptible to iron-related disorders. These tissues take up plasma iron from transferrin or non-transferrin-bound iron, which appears during iron overload. Here, we assessed the effect of iron status on the levels of the transmembrane transporters, ZRT/IRT-like protein 14 and divalent metal-ion transporter-1, which have both been implicated in transferrin- and non-transferrin-bound iron uptake. Weanling male rats (n=6/group) were fed an iron-deficient, iron-adequate, or iron-overloaded diet for 3 weeks. ZRT/IRT-like protein 14, divalent metal-ion transporter-1 protein and mRNA levels in liver, pancreas, and heart were determined by using immunoblotting and quantitative reverse transcriptase polymerase chain reaction analysis. Confocal immunofluorescence microscopy was used to localize ZRT/IRT-like protein 14 in the liver and pancreas. ZRT/IRT-like protein 14 and divalent metal-ion transporter-1 protein levels were also determined in hypotransferrinemic mice with genetic iron overload. Hepatic ZRT/IRT-like protein 14 levels were found to be 100% higher in iron-loaded rats than in iron-adequate controls. By contrast, hepatic divalent metal-ion transporter-1 protein levels were 70% lower in iron-overloaded animals and nearly 3-fold higher in iron-deficient ones. In the pancreas, ZRT/IRT-like protein 14 levels were 50% higher in iron-overloaded rats, and in the heart, divalent metal-ion transporter-1 protein levels were 4-fold higher in iron-deficient animals. At the mRNA level, ZRT/IRT-like protein 14 expression did not vary with iron status, whereas divalent metal-ion transporter-1 expression was found to be elevated in iron-deficient livers. Immunofluorescence staining localized ZRT/IRT-like protein 14 to the basolateral membrane of hepatocytes and to acinar cells of the pancreas. Hepatic ZRT/IRT-like protein 14, but not divalent metal-ion transporter-1, protein levels were elevated in iron-loaded hypotransferrinemic mice. In conclusion, ZRT/IRT-like protein 14 protein levels are up-regulated in iron-loaded rat liver and pancreas and in hypotransferrinemic mouse liver. Divalent metal-ion transporter-1 protein levels are down-regulated in iron-loaded rat liver, and up-regulated in iron-deficient liver and heart. Our results provide insight into the potential contributions of these transporters to tissue iron uptake during iron deficiency and overload. PMID:23349308

Heat shock factor-1 knockout induces multidrug resistance gene, MDR1b, and enhances P-glycoprotein (ABCB1)-based drug extrusion in the heart

PubMed Central

Krishnamurthy, Karthikeyan; Vedam, Kaushik; Kanagasabai, Ragu; Druhan, Lawrence J.; Ilangovan, Govindasamy

2012-01-01

Heat-shock factor 1 (HSF-1), a transcription factor for heat-shock proteins (HSPs), is known to interfere with the transcriptional activity of many oncogenic factors. In the present work, we have discovered that HSF-1 ablation induced the multidrug resistance gene, MDR1b, in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug-resistant cancer cells. The increase in P-gp enhanced the extrusion of doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Dox-induced left ventricular (LV) dysfunction was significantly reduced in HSF-1−/− mice. DNA-binding activity of NF-κB was higher in HSF-1−/− mice. IκB, the NF-κB inhibitor, was depleted due to enhanced IκB kinase (IKK)-α activity. In parallel, MDR1b gene expression and a large increase in P-gp and lowering Dox loading were observed in HSF-1−/− mouse hearts. Moreover, application of the P-gp antagonist, verapamil, increased Dox loading in HSF-1−/− cardiomyocytes, deteriorated cardiac function in HSF-1−/− mice, and decreased survival. MDR1 promoter activity was higher in HSF-1−/− cardiomyocytes, whereas a mutant MDR1 promoter with heat-shock element (HSE) mutation showed increased activity only in HSF-1+/+ cardiomyocytes. However, deletion of HSE and NF-κB binding sites diminished luminescence in both HSF-1+/+ and HSF-1−/− cardiomyocytes, suggesting that HSF-1 inhibits MDR1 activity in the heart. Thus, because high levels of HSF-1 are attributed to poor prognosis of cancer, systemic down-regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapy-induced cardiotoxicity and enhance cancer prognosis. PMID:22615365

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.

PubMed

Federico, Marilen; Portiansky, Enrique L; Sommese, Leandro; Alvarado, Francisco J; Blanco, Paula G; Zanuzzi, Carolina N; Dedman, John; Kaetzel, Marcia; Wehrens, Xander H T; Mattiazzi, Alicia; Palomeque, Julieta

2017-06-15

Spontaneous sarcoplasmic reticulum (SR) Ca 2+ release events increased in fructose-rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca 2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [ 3 H]ryanodine binding and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2-S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD-WT mice, which was prevented by co-treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD-SR-autocamide inhibitory peptide (AIP) mice, expressing the SR-targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD-SR-AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD-WT mice but not in FRD-S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca 2+ /calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca 2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca 2+ spontaneous events in the absence of SR Ca 2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [ 3 H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca 2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favour Ca 2+ transit between the two organelles. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.

PubMed

Rodrigues, Andria V; Kandegedara, Ashoka; Rotondo, John A; Dancis, Andrew; Stemmler, Timothy L

2015-06-01

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

Formulation optimization of aprepitant microemulsion-loaded silicated corn fiber gum particles for enhanced bioavailability.

PubMed

Kamboj, Sunil; Rana, Vikas

2016-08-01

The present investigation was aimed at development of silicate corn fiber gum (SCFG) particles as superior solid carrier for the preparation of Aprepitant (APT)-loaded self-emulsifying powder (SEP) system. 2(4) D-optimal mixture design with three level process variables was employed to develop SCFG particles, utilizing flow descriptors and hydrophobicity descriptors as response variables. The results indicated that blending of CFG (51.4% w/w) and magnesium silicate (MS) (48.6% w/w) using freeze-drying technique was found to have highest desirability (0.904). The developed SEP showed highest oil desorbing capacity, low self-emulsification time and highest drug content. It was observed that SCFG-SEP (F2 formulation) showed lowest PDI (0.2445 ± 0.03) as well as smallest particle size (127 ± 5.8 nm). The droplets were uniform and maintain their integrity after reconstitution (TEM analysis). Furthermore, APT-loaded SEP showed enhanced in vitro dissolution (4 folds) and ex vivo performance (7-fold enhanced Papp) as compared to pure APT. Furthermore, in vivo pharmacokinetic study showed that significant enhancement (p > 0.05) in Cmax was evident with APT-loaded F2 (SCFG-SEP) (1.93-fold) and F4 (Aerosil 200-SEP) (1.58-fold). The data also suggested increase in absorption rate when APT incorporated into SCFG-SEP. Thus, findings pointed toward enhanced bioavailability of APT when loaded into SCFG particles. Overall, the developed SCFG particles could be considered as a better alternative to already available solid carrier(s).

Postteneral Protein Feeding may Improve Biological Control Efficiency of Aphytis lingnanensis and Aphytis melinus

PubMed Central

Vanaclocha, Pilar; Papacek, Dan; Verdú, Maria Jesús; Urbaneja, Alberto

2014-01-01

Abstract The augmentative releases of mass-reared Aphytis spp. (Hymenoptera: Aphelinidae) parasitoids are widely used against armored scales. The nutritional status and the initial egg load of Aphytis spp. females are key to their success as biological control agents. For these reasons, this work focuses on the study of providing a protein feed to Aphytis lingnanensis (Compere) and A. melinus DeBach to improve the egg load before their release. The addition of protein to a honey diet during the first 2 d after the adult parasitoid emergence increased the initial egg load in both species of parasitoids by more than five eggs. Furthermore, the addition of protein increased the total number of eggs laid by A. lingnanensis on oleander scale, Aspidiotus nerii Bouché (Hemiptera: Diaspididae). In contrast, this effect was not observed on A. melinus probably because A. nerii is considered a suboptimal host for this parasitoid. The host-feeding activities of the two Aphytis species were differentially affected by the addition of protein to their diets. These results may have direct implications for augmentative biological control programs, especially during transportation from insectaries to the field, a period of time when parasitoids are deprived of hosts. PMID:25502042

Tensile loading modulates bone marrow stromal cell differentiation and the development of engineered fibrocartilage constructs.

PubMed

Connelly, John T; Vanderploeg, Eric J; Mouw, Janna K; Wilson, Christopher G; Levenston, Marc E

2010-06-01

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

Frequency response of pig intervertebral disc cells subjected to dynamic hydrostatic pressure.

PubMed

Kasra, Mehran; Merryman, W David; Loveless, Kristen N; Goel, Vijay K; Martin, James D; Buckwalter, Joseph A

2006-10-01

The pathogenesis of vibration-induced disorders of intervertebral disc at the cellular level is largely unknown. Dynamic loads with frequencies close to that of the in vivo human spine resonant frequency (4-6 Hz) have a destructive effect, which may induce extracellular disc matrix (ECM) degradation. To investigate this issue, three-dimensional (3D) alginate cultures of normal pig intervertebral disc nucleus and inner annulus cells were tested under dynamic hydrostatic loading. Alginate cultures of each region were divided into six groups; five groups were exposed to cyclic hydrostatic pressures of frequencies 1, 3, 5, 8, and 10 Hz with the same amplitude (1 MPa), and group 6 was the control group (no loading). Cultures of different groups were loaded for 3 days (30 min daily) in a hydraulic chamber. Effects of loading frequency on disc collagen and protein metabolism were investigated by measuring 3H-proline-labeled proteins associated with the cells in the extracellular matrix and release of 3H-proline-labeled molecules into culture medium. The results indicated a poor synthesis rate and more degradation near the 5 Hz frequency. The repeatability of experiments was verified by performing two experiments with the same protocol. Both experiments indicated that a threshold frequency of around 5 Hz disrupted protein metabolism. Copyright (c) 2006 Orthopaedic Research Society.

Nutritional composition, glycemic index, glycemic load, and organoleptical quality of glucomannan-enriched soy milk ice cream

NASA Astrophysics Data System (ADS)

Sa'adah, S.; Candra, O. M.; Nugrahani, G.; Pramono, A.; Afifah, D. N.

2018-01-01

Over the past decades, the number of childhood obesity cases has increased significantly, which led to an increase in the number of adults suffering from degenerative diseases such as diabetes mellitus (DM). Glucomannan-Enriched Soy Milk Ice Cream (GSMIC) may prevent obesity in children. The aim of the study was to test the level of carbohydrates, protein, fat, dietary fiber, glycemic index, glycemic load, and organoleptic quality of GSMIC. This experiment used a completely randomized design to test three formulations of glucomannan flour and soy milk (0.5%, 1.5%, and 2.5%). The products were tested for nutritional composition, and evaluated on glycemic index, glycemic load, and organoleptic quality. GSMIC 2.5% had higher levels of dietary fiber and high carbohydrate, protein, and fat content compared to ice cream (3.99%, 30.7%, 1.50%, 1.33%, respectively). The glycemic index of ice cream and 2.5% GSMIC were 75.83 (75%) and 51.48 (51%), respectively, while the glycemic load of ice cream and 2.5% GSMIC were 9.04 and 11.61, respectively. Based on the organoleptic analysis, formulation preferred by the panellists was 2.5% glucomannan flour. Glucomannan flour affected the level of carbohydrates, protein, fat, dietary fiber, glycemic index, glycemic load, and organoleptic quality in soy milk ice cream.

The Influence of Presentation, Organization, and Example Context on Text Learning

ERIC Educational Resources Information Center

McCrudden, Matthew; Schraw, Gregory; Hartley, Kendall; Kiewra, Kenneth

2004-01-01

This research compared high-load and low-load versions of a text by manipulating text presentation, text organization, and example context on measures of fact and concept learning. The low-load text presentation variable enhanced fact and concept learning and post-reading ease of comprehension ratings. The low-load text organization variable led…

Melter Throughput Enhancements for High-Iron HLW

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruger, A. A.; Gan, Hoa; Joseph, Innocent

2012-12-26

This report describes work performed to develop and test new glass and feed formulations in order to increase glass melting rates in high waste loading glass formulations for HLW with high concentrations of iron. Testing was designed to identify glass and melter feed formulations that optimize waste loading and waste processing rate while meeting all processing and product quality requirements. The work included preparation and characterization of crucible melts to assess melt rate using a vertical gradient furnace system and to develop new formulations with enhanced melt rate. Testing evaluated the effects of waste loading on glass properties and themore » maximum waste loading that can be achieved. The results from crucible-scale testing supported subsequent DuraMelter 100 (DM100) tests designed to examine the effects of enhanced glass and feed formulations on waste processing rate and product quality. The DM100 was selected as the platform for these tests due to its extensive previous use in processing rate determination for various HLW streams and glass compositions.« less

Nandrolone decanoate and load increase remodeling and strength in human supraspinatus bioartificial tendons.

PubMed

Triantafillopoulos, Ioannis K; Banes, Albert J; Bowman, Karl F; Maloney, Melissa; Garrett, William E; Karas, Spero G

2004-06-01

To date, no studies document the effect of anabolic steroids on rotator cuff tendons. Controlled laboratory study. Anabolic steroids enhance remodeling and improve the biomechanical properties of bioartificially engineered human supraspinatus tendons. Bioartificial tendons were treated with either nandrolone decanoate (nonload, steroid, n = 18), loading (load, nonsteroid, n = 18), or both (load, steroid, n = 18). A control group received no treatment (nonload, nonsteroid [NLNS], n = 18). Bioartificial tendons' remodeling was assessed by daily scanning, cytoskeletal organization by staining, matrix metalloproteinase-3 levels by ELISA assay, and biomechanical properties by load-to-failure testing. The load, steroid group showed the greatest remodeling and the best organized actin cytoskeleton. Matrix metallo-proteinase-3 levels in the load, steroid group were greater than those of the nonload, nonsteroid group (P <.05). Ultimate stress and ultimate strain in the load, steroid group were greater than those of the nonload, nonsteroid and nonload, steroid groups (P <.05). The strain energy density in the load, steroid group was greater when compared to other groups (P <.05). Nandrolone decanoate and load acted synergistically to increase matrix remodeling and biomechanical properties of bioartificial tendons. Data suggest anabolic steroids may enhance production of bioartificial tendons and rotator cuff tendon healing in vitro. More research is necessary before such clinical use is recommended.

Organic pollutant loading and biodegradability of firefighting foam

NASA Astrophysics Data System (ADS)

Zhang, Xian-Zhong; Bao, Zhi-ming; Hu, Cheng; Li-Shuai, Jing; Chen, Yang

2017-11-01

Firefighting foam has been widely used as the high-performance extinguishing agent in extinguishing the liquid poor fire. It was concerned for its environmental impacts due to its massive usage. In this study, the organic loading level and the biodegradability of 18 firefighting foams commonly used in China were evaluated and compared. The COD and TOC of firefighting foam concentrates are extremely high. Furthermore, those of foam solutions are also much higher than regular wastewater. The COD/TOC ratio of synthetic foams are higher than protein foams. The 28-day biodegradation rates of 18 firefighting foams are all over 60%, indicating that they are all ready biodegradable. Protein foams (P, FP and FFFP) have the higher organic loading and lower 28-day biodegradation rates compared to the synthetic foams (Class A foam, AFFF and S). The short and long-term impact of protein foams on the environment are larger than synthetic foams.

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

PubMed

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

A transient-enhanced NMOS low dropout voltage regulator with parallel feedback compensation

NASA Astrophysics Data System (ADS)

Han, Wang; Lin, Tan

2016-02-01

This paper presents a transient-enhanced NMOS low-dropout regulator (LDO) for portable applications with parallel feedback compensation. The parallel feedback structure adds a dynamic zero to get an adequate phase margin with a load current variation from 0 to 1 A. A class-AB error amplifier and a fast charging/discharging unit are adopted to enhance the transient performance. The proposed LDO has been implemented in a 0.35 μm BCD process. From experimental results, the regulator can operate with a minimum dropout voltage of 150 mV at a maximum 1 A load and IQ of 165 μA. Under the full range load current step, the voltage undershoot and overshoot of the proposed LDO are reduced to 38 mV and 27 mV respectively.

Training and shape retention in conducting polymer artificial muscles

NASA Astrophysics Data System (ADS)

Tominaga, Kazuo; Hashimoto, Hikaru; Takashima, Wataru; Kaneto, Keiichi

2011-12-01

Electrochemomechanical deformation (ECMD) of the conducting polymer polyaniline film is studied to investigate the behaviour of actuation under tensile loads. The ECMD was induced by the strains due to the insertion of ionic species (cyclic strain) and a creep due to applied loads during the redox cycle. The cyclic strain was enhanced by the experience of high tensile loads, indicating a training effect. The training effect was explained by the enhanced electrochemical activity of the film. The creep was recovered by removal of the tensile load and several electrochemical cycles. This fact indicates that the creep results from the one-dimensional anisotropic deformation, and is retained (shape retention) by the ionic crosslink. The recovery of creep results from the elastic relaxation of the polymer conformation.

Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo.

PubMed

Anand, Preetha; Nair, Hareesh B; Sung, Bokyung; Kunnumakkara, Ajaikumar B; Yadav, Vivek R; Tekmal, Rajeshwar R; Aggarwal, Bharat B

2010-02-01

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with antioxidant, anti-inflammatory, antiproliferative, anticancer, antidiabetic, antirheumatic, and antiviral effects, but its optimum potential is limited by its lack of solubility in aqueous solvents and poor oral bioavailability. We employed a polymer-based nanoparticle approach to improve bioavailability. Curcumin was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy indicated a particle diameter of 80.9 nm. This curcumin, renamed from hereon "as curcumin (NP)", was characterized for its biological activity. In vitro curcumin (NP) exhibited very rapid and more efficient cellular uptake than curcumin. Estrase staining revealed that curcumin (NP) was at least as potent as or more potent than curcumin in inducing apoptosis of leukemic cells and in suppressing proliferation of various tumor cell lines. When examined by electrophoretic gel shift mobility assay, curcumin (NP) was more active than curcumin in inhibiting TNF-induced NF-kappaB activation and in suppression of NF-kappaB-regulated proteins involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). In mice, curcumin (NP) was more bioavailable and had a longer half-life than curcumin. Overall we demonstrate that curcumin-loaded PLGA nanoparticles formulation has enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo over curcumin.

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.

PubMed

Yang, Bin; Wyman, Charles E

2006-07-05

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis. (c) 2006 Wiley Periodicals, Inc.

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

PubMed Central

Chen, Julia C.; Hoey, David A.; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R.

2016-01-01

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitment in vitro. In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cells in vivo. Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitment in vivo and that the primary cilium contributes to this process.—Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708

An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

PubMed

Park, Sang-Hyug; Sim, Woo Young; Park, Sin Wook; Yang, Sang Sik; Choi, Byung Hyune; Park, So Ra; Park, Kwideok; Min, Byoung-Hyun

2006-11-01

In this study, we present a biological micro-electromechanical system and its application to the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs). Actuated by an electromagnetic force, the micro cell exciter was designed to deliver a cyclic compressive load (CCL) with various magnitudes. Two major parts in the system are an actuator and a cartridge-type chamber. The former has a permanent magnet and coil, and the latter is equipped with 7 sample dishes and 7 metal caps. Mixed with a 2.4% alginate solution, the alginate/MSC layers were positioned in the sample dishes; the caps contained chondrogenic defined medium without transforming growth factor-beta (TGF-beta). Once powered, the actuator coil-derived electromagnetic force pulled the metal caps down, compressing the samples. The cyclic load was given at 1-Hz frequency for 10 min twice a day. Samples in the dishes without a cap served as a control. The samples were analyzed at 3, 5, and 7 days after stimulation for cell viability, biochemical assays, histologic features, immunohistochemistry, and gene expression of the chondrogenic markers. Applied to the alginate/MSC layer, the CCL system enhanced the synthesis of cartilage-specific matrix proteins and the chondrogenic markers, such as aggrecan, type II collagen, and Sox9. We found that the micromechanically exerted CCL by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-beta.

PEGylated lipid bilayer-wrapped nano-graphene oxides for synergistic co-delivery of doxorubicin and rapamycin to prevent drug resistance in cancers

NASA Astrophysics Data System (ADS)

Thapa, Raj Kumar; Byeon, Jeong Hoon; Choi, Han-Gon; Yong, Chul Soon; Kim, Jong Oh

2017-07-01

Nano-graphene oxide (nGO) is a carbon allotrope studied for its potential as carrier for chemotherapeutic delivery and its photoablation effects. However, interaction of nGO with blood components and the subsequent toxicities warrant a hybrid system for effective cancer drug delivery. Combination chemotherapy aids in effective cancer treatment and prevention of drug resistance. Therefore, in this study, we attempted to prepare polyethylene glycosylated (PEGylated) lipid bilayer-wrapped nGO co-loaded with doxorubicin (DOX) and rapamycin (RAPA), GOLDR, for the prevention and treatment of resistant cancers. Our results revealed a stable GOLDR formulation with appropriate particle size (∼170 nm), polydispersity (∼0.19) and drug loading. Free drug combination (DOX and RAPA) presented synergistic anticancer effects in MDA-MB-231, MCF-7, and BT474 cells. Treatment with GOLDR formulation maintained this synergism in treated cancer cells, which was further enhanced by the near infrared (NIR) laser irradiation-induced photothermal effects of nGO. Higher chromatin condensation and apoptotic body formation, and enhanced protein expression of apoptosis-related markers (Bax, p53, p21, and c-caspase 3) following GOLDR treatment in the presence of NIR laser treatment clearly suggests its superiority in effective chemo-photothermal therapy of resistant cancers. The hybrid nanosystem that we developed provides a basis for the effective use of GOLDR treatment in the prevention and treatment of resistant cancer types.

Effect of sludge retention time on continuous-flow system with enhanced biological phosphorus removal granules at different COD loading.

PubMed

Li, Dong; Lv, Yufeng; Zeng, Huiping; Zhang, Jie

2016-11-01

The effect of sludge retention time (SRT) on the continuous-flow system with enhanced biological phosphorus removal (EBPR) granules at different COD loading was investigated during the operation of more than 220days. And the results showed that when the system operated at long SRT (30days) and low COD loading (200mg·L(-1)), it could maintain excellent performance. However, long SRT and high COD loading (300mg·L(-1)) deteriorated the settling ability of granules and the performance of system and resulted in the overgrowth of filamentous bacteria. Meanwhile, the transformation of poly-β-hydroxyalkanoates (PHAs) and glycogen in metabolism process was inhibited. Moreover, the results of pyrosequencing indicated that filamentous bacteria had a competitive advantage over polyphosphate-accumulating organisms (PAOs) at high COD loading and long SRT. The PAOs specious of Candidatus_Accumlibater and system performance increased obviously when the SRT was reduced to 20days at high COD loading. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of COD loading on the granule-based enhanced biological phosphorus removal system and the recoverability.

PubMed

Yu, Shenjing; Sun, Peide; Zheng, Wei; Chen, Lujun; Zheng, Xiongliu; Han, Jingyi; Yan, Tao

2014-11-01

In this study, the effect of varied COD loading (200, 400, 500, 600 and 800 mg L(-1)) on stability and recoverability of granule-based enhanced biological phosphorus removal (EBPR) system was investigated during continuously 53-d operation. Results showed that COD loading higher than 500 mg L(-1) could obviously deteriorate the granular EBPR system and result in sludge bulking with filamentous bacteria. High COD loading also changed the transformation patterns of poly-β-hydroxyalkanoates (PHAs) and glycogen in metabolism process of polyphosphate-accumulating organisms (PAOs) and inhibited the EPS secretion, which completely destroyed the stability and integrality of granules. Results of FISH indicated that glycogen-accumulating organisms (GAOs) and other microorganisms had a competitive advantage over PAOs with higher COD loading. The community composition and EBPR performance were recovered irreversibly in long time operation when COD loading was higher than 500 mg L(-1). Copyright © 2014 Elsevier Ltd. All rights reserved.

Co-Expression of Regulator of G Protein Signaling 4 (RGS4) and the MU Opioid Receptor in Regions of Rat Brain: Evidence That RGS4 Attenuates MU Opioid Receptor Signaling

DTIC Science & Technology

2003-01-01

coupled receptor signal transduction proposes that agonist-induced conformational changes in the receptor result in an enhanced release of GDP...Regulators of G protein Signalling (RGS) proteins influence G protein-coupled receptor signal transduction by enhancing the intrinsic GTPase activity...of G proteins. The RGS- enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors

Visualization of a radical B 12 enzyme with its G-protein chaperone

DOE PAGES

Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...

2015-02-09

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

PubMed Central

Nallamsetty, Sreedevi; Waugh, David S.

2007-01-01

Certain highly soluble proteins, such as Escherichia coli maltose-binding protein (MBP), have the ability to enhance the solubility of their fusion partners, making them attractive vehicles for the production of recombinant proteins, yet the mechanism of solubility enhancement remains poorly understood. Here, we report that the solubility-enhancing properties of MBP are dramatically affected by amino acid substitutions that alter the equilibrium between its “open” and “closed” conformations. Our findings indicate that the solubility-enhancing activity of MBP is mediated by its open conformation and point to a likely role for the ligand-binding cleft in the mechanism of solubility enhancement. PMID:17964542

Catalytic ozonation of aqueous phenol over metal-loaded HZSM-5.

PubMed

Amin, Nor Aishah Saidina; Akhtar, Javaid; Rai, H K

2011-01-01

The performances of HZSM-5 and transition metal-loaded HZSM-5 (Mn, Cu, Fe, Ti) catalysts during catalytic ozonation of phenol have been investigated. It was observed the performance order for removal of phenol and COD was Mn/HZSM-5 > Fe/HZSM-5 > Cu/HZSM-5 > Ti/HZSM-5 > HZSM-5. The presence of metals on HZSM-5 enhanced the phenol removal capability of HZSM-5. Mn loading on HZSM-5 was optimized due to its high phenol removal capability amongst metal-loaded HZSM-5 catalysts. Experimental results suggested that low amount of Mn loading on HZSM-5 was sufficient for HZSM-5 to act as catalyst and adsorbent. A maximum of 95.8 wt% phenols and 70.2 wt% COD were removed over 2 wt% Mn/HZSM-5 in 120 min. It was supposed that transition metals mainly acted as ozone decomposers due to their multiple oxidation states that enhanced the ozonation of phenol.

Influence of rhBMP-2 on Guided Bone Regeneration for Placement and Functional Loading of Dental Implants: A Radiographic and Histologic Study in Dogs.

PubMed

Ben Amara, Heithem; Lee, Jung-Won; Kim, Jung-Ju; Kang, Yun-Mi; Kang, Eun-Jung; Koo, Ki-Tae

Evidence on the outcomes of functional loading placed in recombinant human bone morphogenetic protein 2 (rhBMP-2)/acellular collagen sponge (ACS)-induced bone is lacking. The aim of this study was to verify whether guided bone regeneration (GBR) with rhBMP-2/ACS enhances regeneration of missing bone and osseointegration of dental implants subject to functional loading. Two bilateral standardized large saddle-type defects (≈10 × 10 × 6 mm) were surgically created in each mandible of seven beagle dogs 2 months after tooth extraction. Defects were immediately reconstructed randomly using rhBMP-2 (O-BMP or InFuse) soaked in ACS, deproteinized bovine bone mineral (DBBM) granules, or ACS alone as surgical control and subsequently covered with collagen membrane. Screw-type sand-blasted, acid-etched dental implants were placed 3 months later into the reconstructed defects and into adjacent bone. Osseointegration was allowed to progress for 3 months before functional loading of 3 months until sacrifice. Significantly more bone fill was radiographically observed for GBR with rhBMP-2/ACS (O-BMP: 92.5%, InFuse: 79%) in comparison to the DBBM (52%) and ACS alone groups (56.6%). Osseointegration was achieved and maintained in all experimental defects challenged by prostheses-driven functional load. The bone density ranged from 37.49% in the ACS group to 64.9% in the rhBMP-2/ACS (InFuse) group with no significance. The highest mean percentage of BIC was found in rhBMP-2/ACS (InFuse: 52.98%) with no statistical difference. Crestal bone resorption was observed around implants placed in reconstructed areas without any significant difference. GBR with rhBMP-2/ACS provided the greatest bone fill among the three treatment procedures. GBR with rhBMP-2/ACS showed efficacy for placement, osseointegration, and functional loading of titanium implants in alveolar ridge defects.

Minocycline enhances the mesenchymal stromal/stem cell pro-healing phenotype in triple antimicrobial-loaded hydrogels.

PubMed

Guerra, Alberto Daniel; Rose, Warren E; Hematti, Peiman; Kao, W John

2017-03-15

Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties including an anti-inflammatory cytokine profile and the promotion of angiogenesis via expression of growth factors in pre-clinical models. MSCs encapsulated in poly(ethylene glycol) diacrylate (PEGdA) and thiolated gelatin poly(ethylene glycol) (Gel-PEG-Cys) crosslinked hydrogels have led to controlled cellular presentation at wound sites with favorable wound healing outcomes. However, the therapeutic potential of MSC-loaded hydrogels may be limited by non-specific protein adsorption on the delivery matrix that could facilitate the initial adhesion of microorganisms and subsequent virulent biofilm formation. Antimicrobials loaded concurrently in the hydrogels with MSCs could reduce microbial bioburden and promote healing, but the antimicrobial effect on the MSC wound healing capacity and the antibacterial efficacy of the hydrogels is unknown. We demonstrate that minocycline specifically induces a favorable change in MSC migration capacity, proliferation, gene expression, extracellular matrix (ECM) attachment, and adhesion molecule and growth factor release with subsequent increased angiogenesis. We then demonstrate that hydrogels loaded with MSCs, minocycline, vancomycin, and linezolid can significantly decrease bacterial bioburden. Our study suggests that minocycline can serve as a dual mechanism for the regenerative capacity of MSCs and the reduction of bioburden in triple antimicrobial-loaded hydrogels. Wound healing is a complex biological process that can be hindered by bacterial infection, excessive inflammation, and inadequate microvasculature. In this study, we develop a new formulation of poly(ethylene glycol) diacrylate and thiolated gelatin poly(ethylene glycol) crosslinked hydrogels loaded with minocycline, vancomycin, linezolid, and mesenchymal stromal/stem cells that induces a favorable wound healing phenotype in mesenchymal stromal/stem cells and prevents bacterial bioburden on the hydrogel. This combinatorial approach to biomaterial development has the potential to impact wound healing for contaminated full thickness cutaneous wounds. Copyright © 2017. Published by Elsevier Ltd.

Improving the Quality of Online Discussion: The Effects of Strategies Designed Based on Cognitive Load Theory Principles

ERIC Educational Resources Information Center

Darabi, Aubteen; Jin, Li

2013-01-01

This article focuses on heavy cognitive load as the reason for the lack of quality associated with conventional online discussion. Using the principles of cognitive load theory, four online discussion strategies were designed specifically aiming at reducing the discussants' cognitive load and thus enhancing the quality of their online discussion.…

Process analysis and optimization of simultaneous saccharification and co-fermentation of ethylenediamine-pretreated corn stover for ethanol production.

PubMed

Qin, Lei; Zhao, Xiong; Li, Wen-Chao; Zhu, Jia-Qing; Liu, Li; Li, Bing-Zhi; Yuan, Ying-Jin

2018-01-01

Improving ethanol concentration and reducing enzyme dosage are main challenges in bioethanol refinery from lignocellulosic biomass. Ethylenediamine (EDA) pretreatment is a novel method to improve enzymatic digestibility of lignocellulose. In this study, simultaneous saccharification and co-fermentation (SSCF) process using EDA-pretreated corn stover was analyzed and optimized to verify the constraint factors on ethanol production. Highest ethanol concentration was achieved with the following optimized SSCF conditions at 6% glucan loading: 12-h pre-hydrolysis, 34 °C, pH 5.4, and inoculum size of 5 g dry cell/L. As glucan loading increased from 6 to 9%, ethanol concentration increased from 33.8 to 48.0 g/L, while ethanol yield reduced by 7%. Mass balance of SSCF showed that the reduction of ethanol yield with the increasing solid loading was mainly due to the decrease of glucan enzymatic conversion and xylose metabolism of the strain. Tween 20 and BSA increased ethanol concentration through enhancing enzymatic efficiency. The solid-recycled SSCF process reduced enzyme dosage by 40% (from 20 to 12 mg protein/g glucan) to achieve the similar ethanol concentration (~ 40 g/L) comparing to conventional SSCF. Here, we established an efficient SSCF procedure using EDA-pretreated biomass. Glucose enzymatic yield and yeast viability were regarded as the key factors affecting ethanol production at high solid loading. The extensive analysis of SSCF would be constructive to overcome the bottlenecks and improve ethanol production in cellulosic ethanol refinery.

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope.

PubMed

Hufnagl, Karin; Ghosh, Debajyoti; Wagner, Stefanie; Fiocchi, Alessandro; Dahdah, Lamia; Bianchini, Rodolfo; Braun, Nina; Steinborn, Ralf; Hofer, Martin; Blaschitz, Marion; Roth, Georg A; Hofstetter, Gerlinde; Roth-Walter, Franziska; Pacios, Luis F; Jensen-Jarolim, Erika

2018-01-25

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Exosomes as Drug Delivery Vehicles for Parkinson’s Disease Therapy

PubMed Central

Haney, Matthew J.; Klyachko, Natalia L.; Zhao, Yuling; Gupta, Richa; Plotnikova, Evgeniya G.; He, Zhijian; Patel, Tejash; Piroyan, Aleksandr; Sokolsky, Marina; Kabanov, Alexander V.; Batrakova, Elena V.

2015-01-01

Exosomes are naturally occurring nanosized vesicles that have attracted considerable attention as drug delivery vehicles in the past few years. Exosomes are comprised of natural lipid bilayers with the abundance of adhesive proteins that readily interact with cellular membranes. We posit that exosomes secreted by monocytes and macrophages can provide an unprecedented opportunity to avoid entrapment in mononuclear phagocytes (as a part of the host immune system), and at the same time enhance delivery of incorporated drugs to target cells ultimately increasing drug therapeutic efficacy. In light of this, we developed a new exosomal-based delivery system for a potent antioxidant, catalase, to treat Parkinson’s disease (PD). Catalase was loaded into exosomes ex vivo using different methods: the incubation at room temperature, permeabilization with saponin, freeze-thaw cycles, sonication, or extrusion. The size of the obtained catalase-loaded exosomes (exoCAT) was in the range of 100 - 200 nm. A reformation of exosomes upon sonication and extrusion, or permeabilization with saponin resulted in high loading efficiency, sustained release, and catalase preservation against proteases degradation. Exosomes were readily taken up by neuronal cells in vitro. A considerable amount of exosomes was detected in PD mouse brain following intranasal administration. ExoCAT provided significant neuroprotective effects in in vitro and in vivo models of PD. Overall, exosome-based catalase formulations have a potential to be a versatile strategy to treat inflammatory and neurodegenerative disorders. PMID:25836593

Very Low-Protein Diet (VLPD) Reduces Metabolic Acidosis in Subjects with Chronic Kidney Disease: The “Nutritional Light Signal” of the Renal Acid Load

PubMed Central

Di Iorio, Biagio Raffaele; Di Micco, Lucia; Marzocco, Stefania; De Simone, Emanuele; De Blasio, Antonietta; Sirico, Maria Luisa; Nardone, Luca

2017-01-01

Background: Metabolic acidosis is a common complication of chronic kidney disease; current guidelines recommend treatment with alkali if bicarbonate levels are lower than 22 mMol/L. In fact, recent studies have shown that an early administration of alkali reduces progression of CKD. The aim of the study is to evaluate the effect of fruit and vegetables to reduce the acid load in CKD. Methods: We conducted a case-control study in 146 patients who received sodium bicarbonate. Of these, 54 patients assumed very low-protein diet (VLPD) and 92 were controls (ratio 1:2). We calculated every three months the potential renal acid load (PRAL) and the net endogenous acid production (NEAP), inversely correlated with serum bicarbonate levels and representing the non-volatile acid load derived from nutrition. Un-paired T-test and Chi-square test were used to assess differences between study groups at baseline and study completion. Two-tailed probability values ≤0.05 were considered statistically significant. Results: At baseline, there were no statistical differences between the two groups regarding systolic blood pressure (SBP), diastolic blood pressure (DBP), protein and phosphate intake, urinary sodium, potassium, phosphate and urea nitrogen, NEAP, and PRAL. VLPD patients showed at 6 and 12 months a significant reduction of SBP (p < 0.0001), DBP (p < 0.001), plasma urea (p < 0.0001) protein intake (p < 0.0001), calcemia (p < 0.0001), phosphatemia (p < 0.0001), phosphate intake (p < 0.0001), urinary sodium (p < 0.0001), urinary potassium (p < 0.002), and urinary phosphate (p < 0.0001). NEAP and PRAL were significantly reduced in VLPD during follow-up. Conclusion: VLPD reduces intake of acids; nutritional therapy of CKD, that has always taken into consideration a lower protein, salt, and phosphate intake, should be adopted to correct metabolic acidosis, an important target in the treatment of CKD patients. We provide useful indications regarding acid load of food and drinks—the “acid load dietary traffic light”. PMID:28106712

Biophysical Characterization of the Type III Secretion Tip Proteins and the Tip Proteins Attached to Bacterium-Like Particles

PubMed Central

Choudhari, Shyamal P.; Chen, Xiaotong; Kim, Jae Hyun; van Roosmalen, Maarten L.; Greenwood, Jamie C.; Joshi, Sangeeta B.; Picking, William D.; Leenhouts, Kees; Middaugh, C. Russell; Picking, Wendy L.

2014-01-01

Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring domain (PA). In this study, the tip proteins IpaD, SipD and LcrV belonging to type three secretion systems of Shigella flexneri, Salmonella enterica and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation. PMID:24916512

Solvent extraction employing a static micromixer: a simple, robust and versatile technology for the microencapsulation of proteins.

PubMed

Freitas, S; Walz, A; Merkle, H P; Gander, B

2003-01-01

The potential of a static micromixer for the production of protein-loaded biodegradable polymeric microspheres by a modified solvent extraction process was examined. The mixer consists of an array of microchannels and features a simple set-up, consumes only very small space, lacks moving parts and offers simple control of the microsphere size. Scale-up from lab bench to industrial production is easily feasible through parallel installation of a sufficient number of micromixers ('number-up'). Poly(lactic-co-glycolic acid) microspheres loaded with a model protein, bovine serum albumin (BSA), were prepared. The influence of various process and formulation parameters on the characteristics of the microspheres was examined with special focus on particle size distribution. Microspheres with monomodal size distributions having mean diameters of 5-30 micro m were produced with excellent reproducibility. Particle size distributions were largely unaffected by polymer solution concentration, polymer type and nominal BSA load, but depended on the polymer solvent. Moreover, particle mean diameters could be varied in a considerable range by modulating the flow rates of the mixed fluids. BSA encapsulation efficiencies were mostly in the region of 75-85% and product yields ranged from 90-100%. Because of its simple set-up and its suitability for continuous production, static micromixing is suggested for the automated and aseptic production of protein-loaded microspheres.

Utilizing the protein corona around silica nanoparticles for dual drug loading and release

NASA Astrophysics Data System (ADS)

Shahabi, Shakiba; Treccani, Laura; Dringen, Ralf; Rezwan, Kurosch

2015-10-01

A protein corona forms spontaneously around silica nanoparticles (SNPs) in serum-containing media. To test whether this protein corona can be utilized for the loading and release of anticancer drugs we incorporated the hydrophilic doxorubicin, the hydrophobic meloxicam as well as their combination in the corona around SNPs. The application of corona-covered SNPs to osteosarcoma cells revealed that drug-free particles did not affect the cell viability. In contrast, SNPs carrying a protein corona with doxorubicin or meloxicam lowered the cell proliferation in a concentration-dependent manner. In addition, these particles had an even greater antiproliferative potential than the respective concentrations of free drugs. The best antiproliferative effects were observed for SNPs containing both doxorubicin and meloxicam in their corona. Co-localization studies revealed the presence of doxorubicin fluorescence in the nucleus and lysosomes of cells exposed to doxorubicin-containing coated SNPs, suggesting that endocytotic uptake of the SNPs facilitates the cellular accumulation of the drug. Our data demonstrate that the protein corona, which spontaneously forms around nanoparticles, can be efficiently exploited for loading the particles with multiple drugs for therapeutic purposes. As drugs are efficiently released from such particles they may have a great potential for nanomedical applications.A protein corona forms spontaneously around silica nanoparticles (SNPs) in serum-containing media. To test whether this protein corona can be utilized for the loading and release of anticancer drugs we incorporated the hydrophilic doxorubicin, the hydrophobic meloxicam as well as their combination in the corona around SNPs. The application of corona-covered SNPs to osteosarcoma cells revealed that drug-free particles did not affect the cell viability. In contrast, SNPs carrying a protein corona with doxorubicin or meloxicam lowered the cell proliferation in a concentration-dependent manner. In addition, these particles had an even greater antiproliferative potential than the respective concentrations of free drugs. The best antiproliferative effects were observed for SNPs containing both doxorubicin and meloxicam in their corona. Co-localization studies revealed the presence of doxorubicin fluorescence in the nucleus and lysosomes of cells exposed to doxorubicin-containing coated SNPs, suggesting that endocytotic uptake of the SNPs facilitates the cellular accumulation of the drug. Our data demonstrate that the protein corona, which spontaneously forms around nanoparticles, can be efficiently exploited for loading the particles with multiple drugs for therapeutic purposes. As drugs are efficiently released from such particles they may have a great potential for nanomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04726a

Carboxylated mesoporous carbon microparticles as new approach to improve the oral bioavailability of poorly water-soluble carvedilol.

PubMed

Zhang, Yanzhuo; Zhi, Zhizhuang; Li, Xue; Gao, Jian; Song, Yaling

2013-09-15

The main objective of this study was to develop carboxylated ordered mesoporous carbon microparticles (c-MCMs) loaded with a poorly water-soluble drug, intended to be orally administered, able to enhance the drug loading capacity and improve the oral bioavailability. A model drug, carvedilol (CAR), was loaded onto c-MCMs via a procedure involving a combination of adsorption equilibrium and solvent evaporation. The physicochemical properties of the drug-loaded composites were systematically studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM), nitrogen adsorption, powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and HPLC. It was found that c-MCM has a high drug loading level up to 41.6%, and higher than that of the mesoporous silica template. Incorporation of CAR in both drug carriers enhanced the solubility and dissolution rate of the drug, compared to the pure crystalline drug. After loading CAR into c-MCMs, its oral bioavailability was compared with the marketed product in dogs. The results showed that the bioavailability of CAR was improved 179.3% compared with that of the commercial product when c-MCM was used as the drug carrier. We believe that the present study will help in the design of oral drug delivery systems for enhanced oral bioavailability of poorly water-soluble drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

Photocatalytic activity of PANI loaded coordination polymer composite materials: Photoresponse region extension and quantum yields enhancement via the loading of PANI nanofibers on surface of coordination polymer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Zhongping; Qi, Ji; Xu, Xinxin, E-mail: xuxx@mail.neu.edu.cn

2013-09-15

To enhance photocatalytic property of coordination polymer in visible light region, polyaniline (PANI) loaded coordination polymer photocatalyst was synthesized through in-situ chemical oxidation of aniline on the surface of coordination polymer. The photocatalytic activity of PANI loaded coordination polymer composite material for degradation of Rhodamine B (RhB) was investigated. Compared with pure coordination polymer photocatalyst, which can decompose RhB merely under UV light irradiation, PANI loaded coordination polymer photocatalyst displays more excellent photocatalytic activity in visible light region. Furthermore, PANI loaded coordination polymer photocatalyst exhibits outstanding stability during the degradation of RhB. - Graphical abstract: PANI loaded coordination polymer compositemore » material, which displays excellent photocatalytic activity under visible light was firstly synthesized through in-situ chemical oxidation of aniline on surface of coordination polymer. Display Omitted - Highlights: • This PANI loaded coordination polymer composite material represents the first conductive polymer loaded coordination polymer composite material. • PANI/coordination polymer composite material displays more excellent photocatalytic activity for the degradation of MO in visible light region. • The “combination” of coordination polymer and PANI will enable us to design high-activity, high-stability and visible light driven photocatalyst in the future.« less

MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

PubMed

Zhao, Yumei; Migita, Keisuke; Sun, Jing; Katsuragi, Takeshi

2010-04-01

Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

PubMed Central

Rose, Christopher M.; Russell, Jason D.; Ledvina, Aaron R.; McAlister, Graeme C.; Westphall, Michael S.; Griep-Raming, Jens; Schwartz, Jae C.; Coon, Joshua J.; Syka, John E.P.

2013-01-01

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section RF ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately two-fold higher quadrupole field frequency of this cell relative to that of the A-QLT, enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation. PMID:23609185

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

NASA Astrophysics Data System (ADS)

Bahreini, Elham; Aghaiypour, Khosrow; Abbasalipourkabir, Roghayeh; Mokarram, Ali Rezaei; Goodarzi, Mohammad Taghi; Saidijam, Massoud

2014-07-01

This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene ( ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.

Detecting Protein-Glycolipid Interactions Using CaR-ESI-MS and Model Membranes: Comparison of Pre-loaded and Passively Loaded Picodiscs.

PubMed

Li, Jun; Han, Ling; Li, Jianing; Kitova, Elena N; Xiong, Zi Jian; Privé, Gilbert G; Klassen, John S

2018-04-13

Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs ( PL PDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB 5 ). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PL PDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PL PDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB 5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB 5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB 5 measured for pre-loaded PDs and PL PDs prepared from glycolipids extracted from pig and mouse brain revealed that the PL PDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PL PDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS. Graphical Abstract ᅟ.

Detecting Protein-Glycolipid Interactions Using CaR-ESI-MS and Model Membranes: Comparison of Pre-loaded and Passively Loaded Picodiscs

NASA Astrophysics Data System (ADS)

Li, Jun; Han, Ling; Li, Jianing; Kitova, Elena N.; Xiong, Zi Jian; Privé, Gilbert G.; Klassen, John S.

2018-04-01

Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs (PLPDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB5). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PLPDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PLPDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB5 measured for pre-loaded PDs and PLPDs prepared from glycolipids extracted from pig and mouse brain revealed that the PLPDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PLPDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS. [Figure not available: see fulltext.

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.

PubMed

Mitchell, Carter A; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2012-04-17

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

PubMed

Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

2013-01-01

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.

Dietary acid load and chronic kidney disease in elderly adults: Protein and potassium intake.

PubMed

Ko, Byung-Joon; Chang, Yoosoo; Ryu, Seungho; Kim, Eun Mi; Lee, Mi Yeon; Hyun, Young Youl; Lee, Kyu-Beck

2017-01-01

Dietary net endogenous acid production (NEAP), which represents total dietary load of nonvolatile acid, may affect kidney function. Estimated NEAP (eNEAP) is calculated indirectly by the ratio of protein and potassium intake. A few studies are available assessing the association between eNEAP and chronic kidney disease (CKD), and its relation to dietary protein and potassium intake in the elderly. A total 1,369 community-dwelling elderly Koreans in the Kangbuk Samsung Cohort Study (KSCS) were evaluated using a food frequency questionnaire (FFQ) and comprehensive health examination. We evaluated the association between eNEAP and the CKD. We also examined their relation to protein and potassium intake. eNEAP was correlated with potassium intake (r = -0.410, P < 0.001), but was not correlated with protein intake (r = -0.004, P = 0.879). In a full multivariate adjustment for sociodemographic factors, dietary factors, and comorbidities, the participants with higher eNEAP quartiles (Q2, Q3, Q4) had higher odds of CKD compared to the lowest eNEAP quartile (Q1); OR (95% CI) were 1.47 (0.78-2.72), 1.66 (0.85-3.23), and 2.30 (1.16-4.60) respectively (P for trend = 0.019). The odds of CKD decreased for participants with higher potassium intake quartiles (Q2, Q3, Q4) compared to the lowest potassium intake quartile (Q1); OR (95% CI) were 0.52 (0.28-0.95), 0.50 (0.26-0.96), and 0.50 (0.21-0.99) respectively (P for trend = 0.050). Protein intake was not associated with CKD. The association between eNEAP and CKD was similar in subgroup analysis. Dietary acid load was associated with CKD. Among the nutrients related to dietary acid load, potassium intake was negatively associated with CKD, but protein intake was not associated with CKD in elderly adults.

Mechanical loading, damping, and load-driven bone formation in mouse tibiae.

PubMed

Dodge, Todd; Wanis, Mina; Ayoub, Ramez; Zhao, Liming; Watts, Nelson B; Bhattacharya, Amit; Akkus, Ozan; Robling, Alexander; Yokota, Hiroki

2012-10-01

Mechanical loads play a pivotal role in the growth and maintenance of bone and joints. Although loading can activate anabolic genes and induce bone remodeling, damping is essential for preventing traumatic bone injury and fracture. In this study we investigated the damping capacity of bone, joint tissue, muscle, and skin using a mouse hindlimb model of enhanced loading in conjunction with finite element modeling to model bone curvature. Our hypothesis was that loads were primarily absorbed by the joints and muscle tissue, but that bone also contributed to damping through its compression and natural bending. To test this hypothesis, fresh mouse distal lower limb segments were cyclically loaded in axial compression in sequential bouts, with each subsequent bout having less surrounding tissue. A finite element model was generated to model effects of bone curvature in silico. Two damping-related parameters (phase shift angle and energy loss) were determined from the output of the loading experiments. Interestingly, the experimental results revealed that the knee joint contributed to the largest portion of the damping capacity of the limb, and bone itself accounted for approximately 38% of the total phase shift angle. Computational results showed that normal bone curvature enhanced the damping capacity of the bone by approximately 40%, and the damping effect grew at an accelerated pace as curvature was increased. Although structural curvature reduces critical loads for buckling in beam theory, evolution apparently favors maintaining curvature in the tibia. Histomorphometric analysis of the tibia revealed that in response to axial loading, bone formation was significantly enhanced in the regions that were predicted to receive a curvature-induced bending moment. These results suggest that in addition to bone's compressive damping capacity, surrounding tissues, as well as naturally-occurring bone curvature, also contribute to mechanical damping, which may ultimately affect bone remodeling and bone quality. Copyright © 2012 Elsevier Inc. All rights reserved.

Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells1

PubMed Central

Vyas, Jatin M.; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J. Christopher; Van der Veen, Annemarthe G.; Ploegh, Hidde L.

2009-01-01

Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. PMID:17513769

A genetically encoded ratiometric sensor to measure extracellular pH in microdomains bounded by basolateral membranes of epithelial cells.

PubMed

Urra, Javier; Sandoval, Moisés; Cornejo, Isabel; Barros, L Felipe; Sepúlveda, Francisco V; Cid, L Pablo

2008-10-01

Extracellular pH, especially in relatively inaccessible microdomains between cells, affects transport membrane protein activity and might have an intercellular signaling role. We have developed a genetically encoded extracellular pH sensor capable of detecting pH changes in basolateral spaces of epithelial cells. It consists of a chimerical membrane protein displaying concatenated enhanced variants of cyan fluorescence protein (ECFP) and yellow fluorescence protein (EYFP) at the external aspect of the cell surface. The construct, termed pHCECSensor01, was targeted to basolateral membranes of Madin-Darby canine kidney (MDCK) cells by means of a sequence derived from the aquaporin AQP4. The fusion of pH-sensitive EYFP with pH-insensitive ECFP allows ratiometric pH measurements. The titration curve of pHCECSensor01 in vivo had a pK (a) value of 6.5 +/- 0.04. Only minor effects of extracellular chloride on pHCECSensor01 were observed around the physiological concentrations of this anion. In MDCK cells, the sensor was able to detect changes in pH secondary to H(+) efflux into the basolateral spaces elicited by an ammonium prepulse or lactate load. This genetically encoded sensor has the potential to serve as a noninvasive tool for monitoring changes in extracellular pH microdomains in epithelial and other tissues in vivo.

Nitric oxide-mediated suppression of 2,3-bisphosphoglycerate synthesis: therapeutic relevance for environmental hypoxia and sickle cell disease.

PubMed

Bertrand, R

2012-09-01

Though hemoglobin (Hb) is best known for transporting oxygen and metabolic wastes throughout the circulatory system, this erythrocyte protein also acts as a hypoxic sensor, its oxygen saturation dependent on the oxygen partial pressure (pO(2)) which varies throughout the vasculature. The production and transport of the endogenous vasodilator nitric oxide (NO) by Hb is dependent on Hb's oxygen saturation, thereby allowing the protein to auto-regulate blood flow efficiency to meet the relative demands of respiring tissues. Erythrocyte concentrations of 2,3-bisphosphoglycerate (BPG), an enhancer of oxygen off-loading from Hb, is very sensitive to changes in glycolytic rates because its synthesis by BPG synthase is dependent on the availability of the glycolytic intermediate 1,3-bisphosphoglycerate. BPG synthase, as well as some glycolytic enzymes, are also very sensitive to pH changes, and variations in BPG levels have direct consequences on the oxygen off-loading function of Hb. I hypothesize that NO may suppress BPG production by (1) inhibiting glyceraldehyde-3-phosphate dehydrogenase (G3PDH), the most critical glycolytic enzyme for the bioavailability of 1,3-bisphosphoglycerate; and to a lesser extent by (2) associated pH changes in the deoxy-Hb-catalyzed depletion of nitrite, a metabolic reservoir of NO. Both mechanisms are favored in low pO(2) environments where BPG is most needed to maximize oxygen off-loading, indicating that the auto-regulatory link between NO and Hb may have inadvertently linked Hb and BPG synthesis in an unfavorable manner. However, for reasons discussed, NO-mediated suppression of BPG may be advantageous in some circumstances; namely, for individuals living at high altitudes and those with the blood disorder sickle cell anemia. This hypothesis is thus relevant to respiratory health under both normative conditions as well as under hypoxic stress. The potential relevance of the hypothesis to comparative animal physiology and evolutionary biology is also briefly described. Copyright © 2012 Elsevier Ltd. All rights reserved.

Calcium homeostasis and bone metabolic responses to protein diets and energy restriction: a randomized control trial

USDA-ARS?s Scientific Manuscript database

Despite some beneficial effects on bone, high protein diets are conventionally considered a primary dietary risk factor for osteoporosis and bone fracture due to the acid load associated with protein catabolism. To test the hypothesis that high dietary protein diets do not negatively affect calcium ...

Coupling Gd-DTPA with a bispecific, recombinant protein anti-EGFR-iRGD complex improves tumor targeting in MRI

PubMed Central

XIN, XIAOYAN; SHA, HUIZI; SHEN, JINGTAO; ZHANG, BING; ZHU, BIN; LIU, BAORUI

2016-01-01

Recombinant anti-epidermal growth factor receptor-internalizing arginine-glycine-aspartic acid (anti-EGFR single-domain antibody fused with iRGD peptide) protein efficiently targets the EGFR extracellular domain and integrin αvβ/β5, and shows a high penetration into cells. Thus, this protein may improve penetration of conjugated drugs into the deep zone of gastric cancer multicellular 3D spheroids. In the present study, a novel tumor-targeting contrast agent for magnetic resonance imaging (MRI) was developed, by coupling gadolinium-diethylene triamine pentaacetate (Gd-DTPA) with the bispecific recombinant anti-EGFR-iRGD protein. The anti-EGFR-iRGD protein was extracted from Escherichia coli and Gd was loaded onto the recombinant protein by chelation using DTPA anhydride. Single-targeting agent anti-EGFR-DTPA-Gd, which served as the control, was also prepared. The results of the present study showed that anti-EGFR-iRGD-DTPA-Gd exhibited no significant cyto toxicity to human gastric carcinoma cells (BGC-823) under the experimental conditions used. Compared with a conventional contrast agent (Magnevist), anti-EGFR-iRGD-DTPA-Gd showed higher T1 relaxivity (10.157/mM/sec at 3T) and better tumor-targeting ability. In addition, the signal intensity and the area under curve for the enhanced signal time in tumor, in vivo, were stronger than Gd-DTPA alone or the anti-EGFR-Gd control. Thus, Gd-labelled anti-EGFR-iRGD has potential as a tumor-targeting contrast agent for improved MRI. PMID:27035336

The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

PubMed

Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

2010-04-01

We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

Design of Protein-Coated Carbon Nanotubes Loaded with Hydrophobic Drugs through Sacrificial Templating of Mesoporous Silica Shells.

PubMed

Fiegel, Vincent; Harlepp, Sebastien; Begin-Colin, Sylvie; Begin, Dominique; Mertz, Damien

2018-03-26

One key challenge in the fields of nanomedicine and tissue engineering is the design of theranostic nanoplatforms able to monitor their therapeutic effect by imaging. Among current developed nano-objects, carbon nanotubes (CNTs) were found suitable to combine imaging, photothermal therapy, and to be loaded with hydrophobic drugs. However, a main problem is their resulting low hydrophilicity. To face this problem, an innovative method is developed here, which consists in loading the surface of carbon nanotubes (CNTs) with drugs followed by a protein coating around them. The originality of this method relies on first covering CNTs with a sacrificial template mesoporous silica (MS) shell grafted with isobutyramide (IBAM) binders on which a protein nanofilm is strongly adhered through IBAM-mediated physical cross-linking. This concept is first demonstrated without drugs, and is further improved with the suitable loading of hydrophobic drugs, curcumin (CUR) and camptothecin (CPT), which are retained between the CNTs and human serum albumin (HSA) layer. Such novel nanocomposites with favorable photothermal properties are very promising for theranostic systems, drug delivery, and phototherapy applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tensile Loading Modulates Bone Marrow Stromal Cell Differentiation and the Development of Engineered Fibrocartilage Constructs

PubMed Central

Connelly, John T.; Vanderploeg, Eric J.; Mouw, Janna K.; Wilson, Christopher G.

2010-01-01

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1–2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro. PMID:20088686

Load-dependent surface diffusion model for analyzing the kinetics of protein adsorption onto mesoporous materials.

PubMed

Marbán, Gregorio; Ramírez-Montoya, Luis A; García, Héctor; Menéndez, J Ángel; Arenillas, Ana; Montes-Morán, Miguel A

2018-02-01

The adsorption of cytochrome c in water onto organic and carbon xerogels with narrow pore size distributions has been studied by carrying out transient and equilibrium batch adsorption experiments. It was found that equilibrium adsorption exhibits a quasi-Langmuirian behavior (a g coefficient in the Redlich-Peterson isotherms of over 0.95) involving the formation of a monolayer of cyt c with a depth of ∼4nm on the surface of all xerogels for a packing density of the protein inside the pores of 0.29gcm -3 . A load-dependent surface diffusion model (LDSDM) has been developed and numerically solved to fit the experimental kinetic adsorption curves. The results of the LDSDM show better fittings than the standard homogeneous surface diffusion model. The value of the external mass transfer coefficient obtained by numerical optimization confirms that the process is controlled by the intraparticle surface diffusion of cyt c. The surface diffusion coefficients decrease with increasing protein load down to zero for the maximum possible load. The decrease is steeper in the case of the xerogels with the smallest average pore diameter (∼15nm), the limit at which the zero-load diffusion coefficient of cyt c also begins to be negatively affected by interactions with the opposite wall of the pore. Copyright © 2017 Elsevier Inc. All rights reserved.

Load-sharing through elastic micro-motion accelerates bone formation and interbody fusion.

PubMed

Ledet, Eric H; Sanders, Glenn P; DiRisio, Darryl J; Glennon, Joseph C

2018-02-13

Achieving a successful spinal fusion requires the proper biological and biomechanical environment. Optimizing load-sharing in the interbody space can enhance bone formation. For anterior cervical discectomy and fusion (ACDF), loading and motion are largely dictated by the stiffness of the plate, which can facilitate a balance between stability and load-sharing. The advantages of load-sharing may be substantial for patients with comorbidities and in multilevel procedures where pseudarthrosis rates are significant. We aimed to evaluate the efficacy of a novel elastically deformable, continuously load-sharing anterior cervical spinal plate for promotion of bone formation and interbody fusion relative to a translationally dynamic plate. An in vivo animal model was used to evaluate the effects of an elastically deformable spinal plate on bone formation and spine fusion. Fourteen goats underwent an ACDF and received either a translationally dynamic or elastically deformable plate. Animals were followed up until 18 weeks and were evaluated by plain x-ray, computed tomography scan, and undecalcified histology to evaluate the rate and quality of bone formation and interbody fusion. Animals treated with the elastically deformable plate demonstrated statistically significantly superior early bone formation relative to the translationally dynamic plate. Trends in the data from 8 to 18 weeks postoperatively suggest that the elastically deformable implant enhanced bony bridging and fusion, but these enhancements were not statistically significant. Load-sharing through elastic micro-motion accelerates bone formation in the challenging goat ACDF model. The elastically deformable implant used in this study may promote early bony bridging and increased rates of fusion, but future studies will be necessary to comprehensively characterize the advantages of load-sharing through micro-motion. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Con: Higher serum bicarbonate in dialysis patients is protective.

PubMed

Chauveau, Philippe; Rigothier, Claire; Combe, Christian

2016-08-01

Metabolic acidosis is often observed in advanced chronic kidney disease, with deleterious consequences on the nutritional status, bone and mineral status, inflammation and mortality. Through clearance of the daily acid load and a net gain in alkaline buffers, dialysis therapy is aimed at correcting metabolic acidosis. A normal bicarbonate serum concentration is the recommended target in dialysis patients. However, several studies have shown that a mild degree of metabolic acidosis in patients treated with dialysis is associated with better nutritional status, higher protein intake and improved survival. Conversely, a high bicarbonate serum concentration is associated with poor nutritional status and lower survival. It is likely that mild acidosis results from a dietary acid load linked to animal protein intake. In contrast, a high bicarbonate concentration in patients treated with dialysis could result mainly from an insufficient dietary acid load, i.e. low protein intake. Therefore, a high pre-dialysis serum bicarbonate concentration should prompt nephrologists to carry out nutritional investigations to detect insufficient dietary protein intake. In any case, a high bicarbonate concentration should be neither a goal of dialysis therapy nor an index of adequate dialysis, whereas mild acidosis could be considered as an indicator of appropriate protein intake. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Preparation and evaluation of microparticles from thiolated polymers via air jet milling.

PubMed

Hoyer, Herbert; Schlocker, Wolfgang; Krum, Kafedjiiski; Bernkop-Schnürch, Andreas

2008-06-01

Microparticles were formulated by incorporation of the model protein horseradish peroxidase in (thiolated) chitosan and (thiolated) poly(acrylic acid) via co-precipitation. Dried protein/polymer complexes were ground with an air jet mill and resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity, release pattern, swelling behaviour and cytotoxicity. The mean particle size distribution was 0.5-12 microm. Non-porous microparticles with a smooth surface were prepared. Microparticles from (thiolated) chitosan had a positive charge whereas microparticles from (thiolated) poly(acrylic acid) were negatively charged. The maximum protein load for microparticles based on chitosan, chitosan-glutathione (Ch-GSH), poly(acrylic acid) (PAA) and for poly(acrylic acid)-glutathione (PAA-GSH) was 7+/-1%, 11+/-2%, 4+/-0.2% and 7+/-2%, respectively. The release profile of all microparticles followed a first order release kinetic. Chitosan (0.5mg), Ch-GSH, PAA and PAA-GSH particles showed a 31.4-, 13.8-, 54.2- and a 42.2-fold increase in weight, respectively. No significant cytotoxicity could be found. Thiolated microparticles prepared by jet milling technique were shown to be stable and to have controlled drug release characteristics. After further optimizations the preparation method described here might be a useful tool for the production of protein loaded drug delivery systems.

Hormonal modulation of novelty processing in women: Enhanced under working memory load with high dehydroepiandrosterone-sulfate-to-dehydroepiandrosterone ratios.

PubMed

do Vale, Sónia; Selinger, Lenka; Martins, João Martin; Bicho, Manuel; do Carmo, Isabel; Escera, Carles

2016-11-10

Several studies have suggested that dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEAS) may enhance working memory and attention, yet current evidence is still inconclusive. The balance between both forms of the hormone might be crucial regarding the effects that DHEA and DHEAS exert on the central nervous system. To test the hypothesis that higher DHEAS-to-DHEA ratios might enhance working memory and/or involuntary attention, we studied the DHEAS-to-DHEA ratio in relation to involuntary attention and working memory processing by recording the electroencephalogram of 22 young women while performing a working memory load task and a task without working memory load in an audio-visual oddball paradigm. DHEA and DHEAS were measured in saliva before each task. We found that a higher DHEAS-to-DHEA ratio was related to enhanced auditory novelty-P3 amplitudes during performance of the working memory task, indicating an increased processing of the distracter, while on the other hand there was no difference in the processing of the visual target. These results suggest that the balance between DHEAS and DHEA levels modulates involuntary attention during the performance of a task with cognitive load without interfering with the processing of the task-relevant visual stimulus. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Beneficial effects of a high-protein, low-glycemic-load hypocaloric diet in overweight and obese women with polycystic ovary syndrome: a randomized controlled intervention study.

PubMed

Mehrabani, Homeira Hamayeli; Salehpour, Saghar; Amiri, Zohreh; Farahani, Sara Jalali; Meyer, Barbara J; Tahbaz, Farideh

2012-04-01

The recommended composition of a hypocaloric diet for obese women with polycystic ovary syndrome (PCOS) is unclear. The aim of this study was to investigate the effects of a high-protein, low-glycemic-load diet compared with a conventional hypocaloric diet on reproductive hormones, inflammatory markers, lipids, glucose, and insulin levels in obese women with PCOS. A total of 60 overweight and obese women with PCOS who did not use insulin-sensitizing agents were recruited and randomly assigned to 1 of the 2 hypocaloric diet groups for a single-blind clinical trial. The groups included a conventional hypocaloric diet (CHCD) (15% of daily energy from protein) and a modified hypocaloric diet (MHCD) with a high-protein, low-glycemic load (30% of daily energy from protein plus low-glycemic-load foods selected from a list) that was prescribed via counseling visits weekly during 12 weeks of study. Anthropometric assessments and biochemical measurements including reproductive hormones, inflammatory factors, lipids, glucose, and insulin were performed on fasting blood samples at baseline and after 12 weeks of dietary intervention. Weight loss was significant and similar in the 2 groups. Mean of testosterone in the MHCD and CHCD groups decreased from 1.78 ± 0.32 to 1.31 ± 0.26 ng/ml and from 1.51 ± 0.12 to 1.15 ± 0.11 ng/ml, respectively (p < 0.001). Follicle sensitizing hormone (FSH), luteinizing hormone (LH), and blood lipids concentrations were not changed except low-density lipoprotein cholesterol (LDL-C) was reduced by 24.5% ± 12.3% (p < 0.001 for both) after 12 weeks of intervention. MHCD resulted in a significant reduction in insulin level, homeostatic model assessment for insulin resistance (HOMA), and high-sensitivity C- reactive protein (hsCRP) concentration (p < 0.001). In this study both hypocaloric diets significantly led to reduced body weight and androgen levels in these two groups of women with PCOS. The combination of high-protein and low-glycemic-load foods in a modified diet caused a significant increase in insulin sensitivity and a decrease in hsCRP level when compared with a conventional diet.

2D double-layer-tube-shaped structure Bi2S3/ZnS heterojunction with enhanced photocatalytic activities

NASA Astrophysics Data System (ADS)

Gao, Xiaoming; Wang, Zihang; Fu, Feng; Li, Xiang; Li, Wenhong

2015-10-01

Bi2S3/ZnS heterojunction with 2D double-layer-tube-shaped structures was prepared by the facile synthesis method. The corresponding relationship was obtained among loaded content to phase, morphology, and optical absorption property of Bi2S3/ZnS composite. The results shown that Bi2S3 loaded could evidently change the crystallinity of ZnS, enhance the optical absorption ability for visible light of ZnS, and improve the morphologies and microstructure of ZnS. The photocatalytic activities of the Bi2S3/ZnS sample were evaluated for the photodegradation of phenol and desulfurization of thiophene under visible light irradiation. The results showed that Bi2S3 loaded greatly improved the photocatalytic activity of ZnS, and the content of loaded Bi2S3 had an impact on the catalytic activity of ZnS. Moreover, the mechanism of enhanced photocatalytic activity was also investigated by analysis of relative band positions of Bi2S3 and ZnS, and photo-generated hole was main active radicals during photocatalytic oxidation process.

Enhanced ionization efficiency in TIMS analyses of plutonium and americium using porous ion emitters

DOE PAGES

Baruzzini, Matthew L.; Hall, Howard L.; Watrous, Matthew G.; ...

2016-12-05

Investigations of enhanced sample utilization in thermal ionization mass spectrometry (TIMS) using porous ion emitter (PIE) techniques for the analyses of trace quantities of americium and plutonium were performed. Repeat ionization efficiency (i.e., the ratio of ions detected to atoms loaded on the filament) measurements were conducted on sample sizes ranging from 10–100 pg for americium and 1–100 pg for plutonium using PIE and traditional (i.e., a single, zone-refined rhenium, flat filament ribbon with a carbon ionization enhancer) TIMS filament sources. When compared to traditional filaments, PIEs exhibited an average boost in ionization efficiency of ~550% for plutonium and ~1100%more » for americium. A maximum average efficiency of 1.09% was observed at a 1 pg plutonium sample loading using PIEs. Supplementary trials were conducted using newly developed platinum PIEs to analyze 10 pg mass loadings of plutonium. As a result, platinum PIEs exhibited an additional ~134% boost in ion yield over standard PIEs and ~736% over traditional filaments at the same sample loading level.« less

FLORIS 2017

DOE Office of Scientific and Technical Information (OSTI.GOV)

2017-08-04

This code is an enhancement to the existing FLORIS code, SWR 14-20. In particular, this enhancement computes overall thrust and turbulence intensity throughout a wind plant. This information is used to form a description of the fatigue loads experienced throughtout the wind plant. FLORIS has been updated to include an optimization routine that optimizes FLORIS to minimize thrust and turbulence intensity (and therefore loads) across the wind plant. Previously, FLORIS had been designed to optimize power out of a wind plant. However, as turbines age, more wind plant owner/operators are looking for ways to reduce their fatigue loads without sacrificingmore » too much power.« less

Design and Synthesis of Self-Assembled Polymeric Nanoparticles for Cancer Drug Delivery

NASA Astrophysics Data System (ADS)

Logie, Jennifer

Current chemotherapeutics are plagued by poor solubility and selectivity, requiring toxic excipients in formulations and causing a number of dose limiting side effects. Nanoparticle delivery has emerged as a strategy to more effectively deliver chemotherapeutics to the tumour site. Specifically, polymeric micelles enable the solubilization of hydrophobic small molecule drugs within the core and mitigate the necessity of excipients. Notwithstanding the significant progress made in polymeric micelle delivery, translation is limited by poor stability and low drug loading. In this work, a rational design approach is used to chemically modify poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol) (P(LA-co-TMCC)-g-PEG) in order to overcome these limitations and effectively deliver drug to tumours. The PEG density of the polymer system was optimized to enhance the stability of our polymeric micelles. Higher PEG densities permitted the lyophilization of micelles and enhanced the serum stability of the system. To increase the drug loading of our system, we facilitated specific intermolecular interactions within the micelle core. For drugs that form colloidal aggregates, such as pentyl-PABC doxazolidine, polymers were used to stabilize the colloidal core against aggregation and protein adsorption. For more challenging molecules, where self-assembly cannot be controlled, such as docetaxel, we modified the polymeric backbone with a peptide from the binding site of the drug to achieve loadings five times higher than those achieved in conventional micelle systems. This novel docetaxel nanoparticle was assessed in vivo in an orthotopic mouse model of breast cancer, where it showed a wider therapeutic index than the conventional ethanolic polysorbate 80 formulation. The improved tolerability of this formulation enabled higher dosing regimens and led to heightened efficacy and survival in this mouse model. Combined, these studies validated P(LA-co-TMCC)-g-PEG nanoparticles as an effective delivery vehicle for two chemotherapeutics, and presents approaches amenable to the delivery of many other clinically relevant hydrophobic drugs or drug combinations.

Heparin-Poloxamer Thermosensitive Hydrogel Loaded with bFGF and NGF Enhances Peripheral Nerve Regeneration in Diabetic Rats.

PubMed

Li, Rui; Li, Yiyang; Wu, Yanqing; Zhao, Yingzheng; Chen, Huanwen; Yuan, Yuan; Xu, Ke; Zhang, Hongyu; Lu, Yingfeng; Wang, Jian; Li, Xiaokun; Jia, Xiaofeng; Xiao, Jian

2018-06-01

Peripheral nerve injury (PNI) is a major burden to society with limited therapeutic options, and novel biomaterials have great potential for shifting the current paradigm of treatment. With a rising prevalence of chronic illnesses such as diabetes mellitus (DM), treatment of PNI is further complicated, and only few studies have proposed therapies suitable for peripheral nerve regeneration in DM. To provide a supportive environment to restore structure and/or function of nerves in DM, we developed a novel thermo-sensitive heparin-poloxamer (HP) hydrogel co-delivered with basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) in diabetic rats with sciatic nerve crush injury. The delivery vehicle not only had a good affinity for large amounts of growth factors (GFs), but also controlled their release in a steady fashion, preventing degradation in vitro. In vivo, compared with HP hydrogel alone or direct GFs administration, GFs-HP hydrogel treatment is more effective at facilitating Schwann cell (SC) proliferation, leading to an increased expression of nerve associated structural proteins, enhanced axonal regeneration and remyelination, and improved recovery of motor function (all p < 0.05). Our mechanistic investigation also revealed that these neuroprotective and neuroregenerative effects of the GFs-HP hydrogel may be associated with activations of phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt), janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. Our work provides a promising therapy option for peripheral nerve regeneration in patients with DM. Copyright © 2018 Elsevier Ltd. All rights reserved.

An enhanced SOCP-based method for feeder load balancing using the multi-terminal soft open point in active distribution networks

DOE PAGES

Ji, Haoran; Wang, Chengshan; Li, Peng; ...

2017-09-20

The integration of distributed generators (DGs) exacerbates the feeder power flow fluctuation and load unbalanced condition in active distribution networks (ADNs). The unbalanced feeder load causes inefficient use of network assets and network congestion during system operation. The flexible interconnection based on the multi-terminal soft open point (SOP) significantly benefits the operation of ADNs. The multi-terminal SOP, which is a controllable power electronic device installed to replace the normally open point, provides accurate active and reactive power flow control to enable the flexible connection of feeders. An enhanced SOCP-based method for feeder load balancing using the multi-terminal SOP is proposedmore » in this paper. Furthermore, by regulating the operation of the multi-terminal SOP, the proposed method can mitigate the unbalanced condition of feeder load and simultaneously reduce the power losses of ADNs. Then, the original non-convex model is converted into a second-order cone programming (SOCP) model using convex relaxation. In order to tighten the SOCP relaxation and improve the computation efficiency, an enhanced SOCP-based approach is developed to solve the proposed model. Finally, case studies are performed on the modified IEEE 33-node system to verify the effectiveness and efficiency of the proposed method.« less

An enhanced SOCP-based method for feeder load balancing using the multi-terminal soft open point in active distribution networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Haoran; Wang, Chengshan; Li, Peng

The integration of distributed generators (DGs) exacerbates the feeder power flow fluctuation and load unbalanced condition in active distribution networks (ADNs). The unbalanced feeder load causes inefficient use of network assets and network congestion during system operation. The flexible interconnection based on the multi-terminal soft open point (SOP) significantly benefits the operation of ADNs. The multi-terminal SOP, which is a controllable power electronic device installed to replace the normally open point, provides accurate active and reactive power flow control to enable the flexible connection of feeders. An enhanced SOCP-based method for feeder load balancing using the multi-terminal SOP is proposedmore » in this paper. Furthermore, by regulating the operation of the multi-terminal SOP, the proposed method can mitigate the unbalanced condition of feeder load and simultaneously reduce the power losses of ADNs. Then, the original non-convex model is converted into a second-order cone programming (SOCP) model using convex relaxation. In order to tighten the SOCP relaxation and improve the computation efficiency, an enhanced SOCP-based approach is developed to solve the proposed model. Finally, case studies are performed on the modified IEEE 33-node system to verify the effectiveness and efficiency of the proposed method.« less

Nanoparticles Penetrate into the Multicellular Spheroid-on-Chip: Effect of Surface Charge, Protein Corona, and Exterior Flow.

PubMed

Huang, Ke; Boerhan, Rena; Liu, Changming; Jiang, Guoqiang

2017-12-04

Nanoparticles (NPs) are widely studied as tumor targeted vehicles. The penetration of NPs into the tumor is considered as a major barrier for delivery of NPs into tumor cell and a big challenge to translate NPs from lab to the clinic. The objective of this study is to know how the surface charge of NPs, the protein corona surrounding the NPs, and the fluid flow around the tumor surface affect the penetration and accumulation of NPs into the tumor, through in vitro penetration study based on a spheroid-on-chip system. Surface decorated polystyrene (PS) NPs (100 nm) carrying positive and negative surface charge were loaded to the multicellular spheroids under static and flow conditions, in the presence or absence of serum proteins. NP penetration was investigated by confocal laser microscopy scanning followed with quantitative image analysis. The results reveal that negatively charged NPs are attached more on the spheroid surface and easier to penetrate into the spheroids. Protein corona, which is formed surrounding the NPs in the presence of serum protein, changes the surface properties of the NPs, weakens the NP-cell affinity, and, therefore, results in lower NP concentration on the spheroid surface but might facilitate deeper penetration. The exterior fluid flow enhances the interstitial flow into the spheroid, which benefits the penetration but also strips the NPs (especially the NPs with protein corona) on the spheroid surface, which decreases the penetration flux significantly. The maximal penetration was obtained by applying negatively charged NPs without protein corona under the flow condition. We hope the present study will help to understand the spatiotemporal performance of drug delivery NPs and inform the rational design of NPs with highly defined drug accumulation localized at a target site.

Effect of polyethylene glycols on the trans-ungual delivery of terbinafine.

PubMed

Nair, Anroop B; Chakraborty, Bireswar; Murthy, S Narasimha

2010-12-01

Topical nail drug delivery could be improved by identifying potent chemical penetration enhancers. The purpose of this study was to assess the effect of polyethylene glycols (PEGs) on the trans-ungual delivery of terbinafine. In vitro permeation studies were carried out by passive and iontophoresis (0.5 mA/cm2) processes for a period of 1 h using gel formulations containing different molecular weight PEGs (30%w/w). The release of drug from the loaded nail plates and the possible mechanisms for the enhanced delivery was studied. Passive delivery using formulation with low molecular weight PEGs (200 and 400 MW) indicated moderate enhancement in the permeation and drug load in the nail plate, compared to the control formulation. However, the effect of low molecular weight PEGs was predominant during iontophoresis process with greater amount of terbinafine being permeated (≈35 µg/cm2) and loaded into the nail plate (≈2.7 µg/mg). However, little or no effect on drug delivery was observed with high molecular weight PEGs (1000- 3350 MW) in passive and iontophoresis processes. Release of drug from the nail plates loaded by iontophoresis using low molecular weight PEG (400 MW) exhibited sustain effect which continued over a period of 72 days. The enhancement in drug permeation by low molecular weight PEGs is likely due to their ability to lead to greater water uptake and swelling of nail. This study concluded that the low molecular weight PEGs are indeed a promising trans-ungual permeation enhancer.

Mapping cavitation activity around dental ultrasonic tips.

PubMed

Walmsley, A Damien; Lea, Simon C; Felver, Bernhard; King, David C; Price, Gareth J

2013-05-01

Cavitation arising within the water around the oscillating ultrasonic scaler tip is an area that may lead to advances in enhancing biofilm removal. The aim of this study is to map the occurrence of cavitation around scaler tips under loaded conditions. Two designs of piezoelectric ultrasonic scaling probes were evaluated with a scanning laser vibrometer and luminol dosimetric system under loaded (100 g/200 g) and unloaded conditions. Loads were applied to the probe tips via teeth mounted in a load-measuring apparatus. There was a positive correlation between probe displacement amplitude and cavitation production for ultrasonic probes. The position of cavitation at the tip of each probe was greater under loaded conditions than unloaded and for the longer P probe towards the tip. Whilst increasing vibration displacement amplitude of ultrasonic scalers increases the occurrence of cavitation, factors such as the length of the probe influence the amount of cavitation activity generated. The application of load affects the production of cavitation at the most clinically relevant area-the tip. Loading and the design of ultrasonic scalers lead to maximising the occurrence of the cavitation at the tip and enhance the cleaning efficiency of the scaler.

Reduction of greenhouse gases by fiber-loaded lightweight, high-opacity newsprint production

Treesearch

John H. Klungness; Matthew L. Stroika; Said M. Abubakr

1999-01-01

We estimated the effectiveness of fiber loading in reducing greenhouse gas emissions for producing lightweight high-opacity newsprint. Fiber loading enhances fiber bonding at increased precipitated calcium carbonate levels without significant loss in Canadian Standard Freeness or additional energy use. We investigated the reduction of greenhouse gas emissions for a...

Gum tragacanth stabilized green gold nanoparticles as cargos for Naringin loading: A morphological investigation through AFM.

PubMed

Rao, Komal; Imran, Muhammad; Jabri, Tooba; Ali, Imdad; Perveen, Samina; Shafiullah; Ahmed, Shakil; Shah, Muhammad Raza

2017-10-15

Gold nanoparticles (AuNPs) have attracted greater scientific interests for the construction of drugs loading cargos due to their biocompatibility, safety and facile surface modifications. This study deals with the fabrication of gum tragacanth (GT) green AuNPs as carrier for Naringin, a less water soluble therapeutic molecule. The optimized AuNPs were characterized through UV-vis spectroscopy, FT-IR and atomic force microscope (AFM). Naringin loaded nanoparticles were investigated for their bactericidal potentials using Tetrazolium Microplate assay. Morphological studies conducted via AFM revealed spherical shape for AuNPs with nano-range size and stabilized by GT multi-functional groups. The AuNPs acted as carrier for increased amount of Naringin. Upon loading in AuNPs, Naringin An increased in the bactericidal potentials of Naringin was observed after loading on AuNPs against various tested bacterial strains. This was further authenticated by the surface morphological analysis, showing enhanced membrane destabilizing effects of loaded Naringin. The results suggest that GT stabilized green AuNPs can act as effective delivery vehicles for enhancing bactericidal potentials of Naringin. Copyright © 2017 Elsevier Ltd. All rights reserved.