Fetal Bovine Serum Solution Research Articles

Solution Behavior of Hydrolyzed Gradient Methyl/Phenyl Oxazoline Copolymers and Complexation with DNA

Partial hydrolysis of 2‐methyl‐2‐oxazoline/2‐phenyl‐2‐oxazoline gradient copolymers (MPOx copolymers) results in amphiphilic copolymers containing cationic ethylene imine, neutral hydrophilic 2‐methyl‐2‐oxazoline, and hydrophobic 2‐phenyl‐2‐oxazoline segments (HMPOx copolymers). The solution behavior of the HMPOx copolymers has been investigated in water, phosphate buffer saline (PBS), and fetal bovine serum (FBS) by static, dynamic, and electrophoretic light scattering and fluorescence spectroscopy. The copolymers form stable spherical aggregates in aqueous media with positive surface charge. The aggregates are found to interact extensively with proteins in FBS solutions. Soluble HMPOx/DNA complexes have also been formed in aqueous media at certain HMPOx/DNA compositions due to electrostatic interactions. The polyplexes have been studied in terms of structure and colloidal stability. image

Impact of Storage Solution Formulation during Refrigerated Storage upon Chondrocyte Viability and Cartilage Matrix

Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzer's and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzer's and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

A simple spectroscopic method for the quantification of gold nanoparticle number concentration in water and fetal bovine serum solutions

In this work, we developed a facile and low-cost method with sufficient sensitivity for the quantitative determination of gold nanoparticles (GNPs) number concentration in water and fetal bovine serum solutions. The Au(III) digested from GNPs was reduced to Au(I) presumably by the thiol group of 2-mercaptobenzimidazole, then the Au(I)–thiolate complex was formed, the concentration of which is determined by absorbance at 300 nm. The number concentration of GNPs can be converted using microscopic images and simple calculations. The interference of serum can be avoided in this method. The limit of detection (LOD) of GNPs was calculated as 2.1 × 10−12 mol/L in water solution and 1.3 × 10−11 mol/L in fetal bovine serum (FBS) solution. This method provides a simple and fast way to determine the concentration of GNPs, which can be in carried out in the routine laboratory practice.

Protein corona on magnetite nanoparticles and internalization of nanoparticle–protein complexes into healthy and cancer cells

Superparamagnetic magnetite nanoparticles (MNPs) of different surface properties are incubated in complicated living fluid, including fetal bovine serum solution, cell complete culture medium and cell culture system with/without serum, to investigate the alteration of protein corona and its impact on cell internalization. The MNPs prepared by co-precipitation method are functionalized with L-Lysine (Lys), Glucosamic acid (GA) to obtain amine, carboxyl and hydroxyl groups, separately. All the particles adsorb serum proteins to form MNPs-protein complexes with the surface charge changing into negative. 1D SDS/PAGE gel images analysis indicates that the composition and content of hard protein corona on the surface of NPs are related to their functional groups and agglomeration, and the total amount of protein in the medium. In cell culture system, particles not only adsorb serum proteins, but also associate with cytosolic proteins arising from HepG2 and L02 cells. GA modified MNPs (MNPs-GA) exhibit bovine serum albumin anti-adsorption capability because of the terminal hydroxyl and carboxyl groups. MNPs-GA also shows the highest cellular uptake and label efficiency compared with uncoated MNPs and Lys modified MNPs, due to larger aggregates formation and specific protein corona composition, rather than commonly approved electrostatic interaction between particles and cells. For the first time, our results provide visualized reports on previously neglected, but indispensable protein corona of the MNPs after interaction with both healthy and cancer cells, suggesting that cytosolic protein corona from cells and aggregation of particles are important factors needed to be account for on studying the nano-bio interface.

Activation of the Leukotriene B4 Receptor 2-Reactive Oxygen Species (BLT2-ROS) Cascade following Detachment Confers Anoikis Resistance in Prostate Cancer Cells

The majority of prostate cancer-related deaths are associated with advanced and metastatic malignancies. Although anoikis resistance has been recognized as one of the hallmarks of metastatic prostate malignancies, the molecular events that cause anoikis resistance are poorly understood. In this study, we found that the detachment of PC-3 prostate cancer cells caused a time-dependent increase in the expression level of the leukotriene B4 receptor-2 (BLT2) and that BLT2 played a critical role in establishing anoikis resistance in these cells. Blocking BLT2 with the pharmacological inhibitor LY255283 or with RNAi knockdown clearly abolished anoikis resistance and resulted in severe apoptotic death. Additionally, we demonstrated that the activation of NADPH oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) were downstream of BLT2 signaling and led to the activation of NF-κB, thus establishing anoikis resistance during cell detachment. Furthermore, we observed that the ectopic expression of BLT2 in normal prostate PWR-1E cells rendered the cells resistant to anoikis and apparently diminished apoptotic cell death following detachment. Taken together, our results suggest that BLT2-NOX-ROS-NF-κB cascade induction during detachment confers a novel mechanism of anoikis resistance in prostate cancer cells and potentially contributes to prostate cancer progression.

The effect of mannosylation of liposome-encapsulated indocyanine green on imaging of sentinel lymph node

The imaging of sentinel lymph nodes (SLN) has been researched for its role in assessing cancer progression and postsurgical lymphedema. Indocyanine green (ICG) is a near-infrared (NIR) optical dye that has been approved by the Food and Drug Administration. It is known that liposome-encapsulated ICG (LP-ICG) has improved stability and fluorescence signal compared with ICG. We designed mannosylated liposome-encapsulated ICG (M-LP-ICG) as an optical contrast agent for SLN. M-LP-ICG has a higher UV absorbance spectrum and fluorescence intensity than LP-ICG. The stability of M-LP-ICG measured in 50% fetal bovine serum solution by a dialysis method was better than that of LP-ICG. M-LP-ICG demonstrated a high uptake in RAW 264.7 macrophage cell because the density of mannose is high. There were differences between M-LP-ICG and glucosylated liposome-encapsulated ICG (G-LP-ICG), which are geometrical isomers. The result of an inhibition study of M-LP-ICG showed a statistically significant decrease in uptake in RAW 264.7 cells after either co-treatment or pre-treatment with d-(+)-mannose as an inhibitor. Results from an in vitro experiment demonstrated that M-LP-ICG was specifically taken up by macrophage cells through the mannose receptor on its surface. The time-series images acquired from a normal mouse model after subcutaneous injection showed that the signal from M-LP-ICG in SLN and other organs appeared early and disappeared quickly in comparison with signals from LP-ICG. Not only the sentinel but also the draining lymph nodes were observed partly in M-LP-ICG. M-LP-ICG appears to increase the specificity of uptake and retention in macrophages, making it a good candidate contrast agent for an optic imaging system for SLN and the lymphatic system.

Engineered Iron-Oxide-Based Nanoparticles as Enhanced T1 Contrast Agents for Efficient Tumor Imaging

We report the design and synthesis of small-sized zwitterion-coated gadolinium-embedded iron oxide (GdIO) nanoparticles, which exhibit a strong T1 contrast effect for tumor imaging through enhanced permeation and retention effect and the ability to clear out of the body in living subjects. The combination of spin-canting effects and the collection of gadolinium species within small-sized GdIO nanoparticles led to a significantly enhanced T1 contrast effect. For example, GdIO nanoparticles with a diameter of ∼4.8 nm exhibited a high r1 relaxivity of 7.85 mM(-1)·S(-1) and a low r2/r1 ratio of 5.24. After being coated with zwitterionic dopamine sulfonate molecules, the 4.8 nm GdIO nanoparticles showed a steady hydrodynamic diameter (∼5.2 nm) in both PBS buffer and fetal bovine serum solution, indicating a low nonspecific protein absorption. This study provides a valuable strategy for the design of highly sensitive iron-oxide-based T1 contrast agents with relatively long circulation half-lives (∼50 min), efficient tumor passive targeting (SKOV3, human ovarian cancer xenograft tumor as a model), and the possibility of rapid renal clearance after tumor imaging.

A new fluorescence “switch on” assay for heparin detection by using a functional ruthenium polypyridyl complex

In the present study, a new strategy for heparin detection and quantification in biological media, such as fetal bovine serum (FBS), is developed by monitoring the emission change of a functional ruthenium polypyridyl complex ([Ru(phen)(2)dppz-idzo](2+), complex 1) in buffer solution. Polyanionic heparin is found to interact with a positively charged Ru-complex through electrostatic effects and/or hydrogen bonding interactions, which leads to a significant fluorescence enhancement of the Ru-complex. To get insight into this fluorescence "switch on" behavior, the binding model of the Ru-complex to heparin is established by employing molecular docking simulations based on the fluorescence and UV absorption results. The selectivity results of the fluorescence assay reveal that our complex displayed good fluorescence selectivity towards heparin over its analogues, such as chondroitin 4-sulfate (Chs) or hyaluronic acid (Hya), which have lower charge density and/or structural compatibility as compared to that of heparin. Quantification of heparin is also performed and a linear calibration curve is observed in the range of 0.01-4.87 U mL(-1) (the limit of detection is 0.01 U mL(-1)) for heparin detection in diluted FBS solution. This "one-step" fluorescence "switch on" assay for heparin detection is label-free, convenient, sensitive and selective, and has a long emission wavelength and large Stokes shift.

Relative binding affinity of carboxylate-, phosphonate-, and bisphosphonate-functionalized gold nanoparticles targeted to damaged bone tissue

Functionalized Au NPs have received considerable recent interest for targeting and labeling cells and tissues. Damaged bone tissue can be targeted by functionalizing Au NPs with molecules exhibiting affinity for calcium. Therefore, the relative binding affinity of Au NPs surface functionalized with either carboxylate (l-glutamic acid), phosphonate (2-aminoethylphosphonic acid), or bisphosphonate (alendronate) was investigated for targeted labeling of damaged bone tissue in vitro. Targeted labeling of damaged bone tissue was qualitatively verified by visual observation and backscattered electron microscopy, and quantitatively measured by the surface density of Au NPs using field-emission scanning electron microscopy. The surface density of functionalized Au NPs was significantly greater within damaged tissue compared to undamaged tissue for each functional group. Bisphosphonate-functionalized Au NPs exhibited a greater surface density labeling damaged tissue compared to glutamic acid- and phosphonic acid-functionalized Au NPs, which was consistent with the results of previous work comparing the binding affinity of the same functionalized Au NPs to synthetic hydroxyapatite crystals. Targeted labeling was enabled not only by the functional groups but also by the colloidal stability in solution. Functionalized Au NPs were stabilized by the presence of the functional groups, and were shown to remain well dispersed in ionic (phosphate buffered saline) and serum (fetal bovine serum) solutions for up to 1 week. Therefore, the results of this study suggest that bisphosphonate-functionalized Au NPs have potential for targeted delivery to damaged bone tissue in vitro and provide motivation for in vivo investigation.

Role of trapped air in the formation of cell-and-protein micropatterns on superhydrophobic/superhydrophilic microtemplated surfaces

Air trapped within the interstices of TiO2 nanotube surfaces bearing superhydrophobic/superhydrophilic microtemplated domains controls formation of protein micropatterns but not cell micropatterns. Protein binding from either bovine-serum albumin (BSA) or fetal-bovine serum (FBS) solutions to superhydrophobic domains is blocked in the presence of trapped air, leading to clear protein binding contrast between superhydrophilic and superhydrophobic domains. Protein binds to superhydrophobic domains when air is displaced by sonication, leading to more protein binding to superhydrophobic domains than to superhydrophilic, with concomitantly blurred protein binding contrast. The overall contrast obtained in formation of cell (hFOB1.19, MG63, and HeLa) micropatterns depends on the cell type and protein composition of the fluid phase. All cell types preferentially attach to superhydrophilic domains from each fluid phase tested (FBS, BSA, and basal media containing no protein). All cell types do not attach to superhydrophobic domains from FBS solutions, with-or-without trapped air, creating a visually-obvious cell attachment pattern. However, cells attached to superhydrophobic domains from basal media suspensions, with-or-without trapped air, creating a blurred cell attachment pattern. Cell attachment from BSA-containing solutions gave mixed results depending on cell type. Thus, trapped air does not necessarily block cell attachment as has been suggested in the literature. Rather, cell attachment is controlled by interfacial tensions between cells, surfaces, and fluid phases in a manner that can be understood in terms of the Dupré work-of-adhesion formulation. Cell attachment patterns developed within the initial attachment phase persist for up to two days of continuous culture but overgrow thereafter, with-or-without trapped air, showing that trapped air does not block cell overgrowth over time of continuous culture.

Mesenchymal stromal cell cryopreservation.

The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

Therapeutical Effect of Extracts of Terminalia chebula In Inhibiting Human Pathogens and Free Radicals

In recent years, multiple drug resistance has been developed due to indiscriminate use of existing antimicrobial drugs in the treatment of infectious diseases. Conventional antibiotics are strong medicines, which if not used in precise way may cause harmful effects. The major thrust is to establish alternative antimicrobial agent in order to treat microbial infections with less or no toxic effect to body and less negligible side effects. The herbal medicines have shown potential to overcome the limitation associated with conventional drugs. However, the appropriate choice of source for herbal medicine is also very important. Terminalia chebula possesses potential pharmaceutical activities and used in several Ayurvedic formulations. The study of solvent-free organic (ethylacetate, acetone, methanol of increasing polarity) and aqueous extracts of the fruits of T. chebula, showed the potential to reduce growth of microorganisms, minimizing the risk of infection, while optimizing the conditions to encourage healing. In our study we found, methanol extract as a potential bactericidal and potent antioxidant while aqueous extract showed the least potential as an antimicrobial agent, though a moderate antioxidant. The finding may provide scientific rationale for the use of crude extract of the plant as a new drug compound as potential antioxidant and antimicrobial agent. Terminalia chebula is used as a mild laxative and as an astringent against wounds and abscesses. Practictioners of folk medicine in India and Southeast Asia use the fruit for homeostatic, laxative and as cardiotonic. It is used as a remedy against a sore throat and cough, against diarrhoea connected with a prolapsed rectum in China and against ulcers and dysentery in Tibet. The antibacterial activity of various extracts of Chebula myrobalan powder was tested against Streptococcus species, Staphylococcus aureus and Pseudomonas aeruginosa. II. MATERIALS T. chebula powder was purchased from local market of Mumbai, India. Pathogens of MTCC grade were obtained from IMTECH (Chandigarh, India). Gallic acid and Glutaraldehyde was purchased from s.d. fine-chemicals limited (Mumbai, India). Gentamicin and 2,4,6-tripyidyl-s-triazine (TPTZ) were procured from Hi-Media(Mumbai,India), 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT, 98%) reagent, 4'-6-diamidino-2-phenylindole (DAPI), propidium iodide (PI) and nystatin was purchased from Sigma (St. Louis, USA). Fetal bovine serum (FBS) and streptomycin-penicillin antibiotic solutions were bought from HyClone (Utah, USA). All other chemicals used were of analytical grade. SIRC (rabbit corneal epithelial cells) cell line was obtained from NCL, Pune, India

Influence of silver content on the tribomechanical behavior on Ag-TiCN bioactive coatings

Surface modification of bulk materials used in biomedical applications has become an important prerequisite for better biocompatibility. In particular, to overcome the particle generation, low-wear coatings based on carbon (nitrogen) and containing antimicrobial elements such as silver are promising candidates. Thus, the present work explores the potentialities of silver-containing carbonitride-based (Ag-TiCN) thin films prepared by direct current unbalanced reactive magnetron sputtering. The silver content in the coatings was varied from 0 to 26.7 at.% by changing the targets and the fraction of C 2H 2 and N 2 in the gas mixture with Ar. The obtained Ag-TiCN based coatings were characterized in terms of composition and microstructure. Mechanical and tribological properties of the films were studied by nanoindentation and reciprocating pin-on disk testing in a fetal bovine serum solution, respectively. Raman, scanning electron microscope and energy dispersive X-ray analysis was carried out in the contact region after tribological tests to obtain information about the friction mechanism. The cytotoxicity of the coatings was assessed by in vitro tests using fibroblast cells. The coatings comprised a mixture of TiC xN 1 − x , Ag and a-C(N) x phases whose relative proportion varied depending on the Ag/Ti ratio. The mechanical, tribological and cytotoxicity properties were correlated with the chemical and phase composition. When the Ag/Ti ratios were below 0.20 (Ag contents < 6.3 at.%) the films resulted harder (~ 18 GPa) with higher wear resistance (~ 10 −6 mm 3/Nm), showing similar friction coefficient (~ 0.3) and good biocompatibility.

Synthesis of lipids for development of multifunctional lipid-based drug-carriers

A simple approach to synthesize phospholipids to modulate drug release and track lipid-based particulate drug-carriers is described. We synthesized two ether lipids, 1 1-O-hexadecyl-2-pentadenoyl-sn-glycerol-3-phosphocholine (C(31)PC) and 2 1-O-hexadecyl-2-pentadenoyl-sn-glycerol-3-phosphomethanol (C(31)PM), and examined their ability to alter enzymatically triggered release of 6-carboxyfluorescein from liposomes incubated in TRIS buffer or fetal bovine serum solutions. Further, we demonstrated that odd-chain lipids, for example, C(31)PC, could be identified in rat plasma without interference of endogenous lipids. This approach can be adapted to synthesize a variety of lipids for use in developing and optimizing multifunctional drug-carriers.

Biocompatible, Antifouling, and Thermosensitive Core−Shell Nanogels Synthesized by RAFT Aqueous Dispersion Polymerization

Reversible addition−fragmentation chain transfer (RAFT) aqueous dispersion polymerization was used to synthesize a novel type of core−shell nanogel containing linear poly(ethylene glycol) (PEG) and/or nonlinear polymers with oligo(ethylene glycol) side chains. These nanogels with low polydispersities were synthesized efficiently with tunable sizes and thermosensitivities. The nanogels containing nonlinear polymers with oligo(ethylene glycol) side chains as the shell had enhanced stability during freeze−thawing process and in biologically relevant solutions including 1.5 M NaCl, 1% bovine serum albumin (BSA) and 100% fetal bovine serum (FBS) solutions. Aminolysis and hydrolysis of the chain transfer agents (CTAs) in the nanogels were studied and the nanogels exhibited enhanced stability in comparison with molecularly dissolved polymers. The chemical stability of the CTAs in the nanogels was well-correlated with the in vitro cell viability studies of the nanogels using lung cancer cells.

Near-infrared (1550 nm) in vivo bioimaging based on rare-earth doped ceramic nanophosphors modified with PEG-b-poly(4-vinylbenzylphosphonate)

A novel poly(ethylene glycol) (PEG)-based block copolymer possessing a 4-vinylbenzylphosphonate repeating unit in another segment (PEG-block-poly(4-vinylbenzylphosphonate)) (PEG-b-PVBP) was designed and successfully synthesized. As a control, an end-functionalized PEG possessing a mono-phosphonate group (PEG-PO(3)H(2)) was also synthesized. The surface of near-infrared (NIR) phosphors (i.e., ytterbium (Yb) and erbium (Er) ion-codoped Y(2)O(3) nanoparticles (YNPs)) were modified with PEG-b-PVBP (PEG-YNP(b)s) and PEG-PO(3)H(2) (PEG-YNP(1)s). The adsorption of PEG-b-PVBP and PEG-PO(3)H(2) was estimated by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The physicochemical characteristics of the obtained YNP samples were analyzed by ζ-potential and dynamic light scattering (DLS) measurements. The ζ-potentials of YNPs modified by these polymers were close to zero, indicating the effective coverage of the YNP surface by our new PEG derivatives. However, the dispersion stability of the PEGylated YNPs was strongly affected by the structure of the PEG terminus. The average diameter of the PEG-YNP(1)s increased, and aggregates precipitated after less than 1 h in phosphate buffer saline (PBS). In contrast, the size did not change at all in the case of PEG-YNP(b)s and the dispersion in PBS was stable for over 1 week. PEG-YNP(b)s also showed high erosion resistance under acidic conditions. The multiple coordinated PVBP segment of the block copolymer on the YNP surface plays a substantial role in improving such dispersion stability. The excellent dispersion stability and strong NIR luminescence of the obtained PEG-YNP(b)s were also confirmed in fetal bovine serum (FBS) solution over 1 week. Furthermore, in vivo NIR imaging of live mice was performed, and the 1550 nm NIR emission of PEG-YNP(b)s from the organ of live mice was confirmed without dissection.

Nucleolar Follistatin Promotes Cancer Cell Survival under Glucose-deprived Conditions through Inhibiting Cellular rRNA Synthesis

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Effect of bovine serum constituents on the surface of the tribological pair alumina/alumina nanocomposites for total hip replacement

Alumina has been widely used in the fabrication of hip joint replacement due to its excellent properties. But one of its drawbacks is the low fracture toughness, which can be enhanced with the addition of a second ceramic phase of nanometer size. Another property to be improved is its wear resistance under severe rubbing conditions. In this work Yttria Tetragonal Zirconia Polycrystal, Y-TZP (TZ-8YS, 100 nm), TiO 2 (PS-25, 50 nm) and Co (Nilaco, 28 nm) were added to Al 2O 3 (AKP 50, 300 nm). These starting powders were mixed and hot pressed, in order to fabricate alumina nanocomposites. Mechanical properties of the nanocomposites were estimated using the indentation method. Wear tests were carried out using a ball-on-plate tribometer, with a frequency of 1 Hz, at a load of 49 N, for 1 h. Alumina balls were used as the counterface. Distilled water and fetal bovine serum solution (FBSS) were the environments. The specific wear rate was within 10 −8 to 10 −7 mm 3/N m and the coefficient of friction was around 0.3–0.7. Keeping in mind that wear is a process that starts on the surface and it is related to the physical chemistry of the surfaces, techniques such as scanning electron microscopy (SEM), and X-ray photoelectron spectrometry (XPS) were used to elucidate the role played by the proteins and phospholipids present in the fetal bovine serum solution and the new chemical specie formed on the surface.

Antibacterial properties of nanostructured silver titanate thin films formed on a titanium plate

Antibacterial activity of various surfaces against methicillin-resistant Staphylococcus aureus (MRSA) was studied. Sodium titanate thin film with a porous network structure and sodium titanate nanotube thin film were formed on titanium surfaces through the reaction of titanium plates with NaOH solutions. Through a silver ion-exchange treatment, Na(+) ions in sodium titanate were exchanged with Ag(+) ions in silver acetate solution, along with the loading of silver nanoparticles on the titanate surfaces. Results of silver ion elution tests of the thin films in fetal bovine serum solution indicate that the release period and the number of silver ions released from the silver titanate thin films can be controlled by altering the crystal structure, nanostructure, and thickness of the titanate phase. The silver ion-exchanged titanate thin films showed high antibacterial activity against MRSA. It was also revealed that although the crystal structure of titanate itself has no large antibacterial effect, higher antibacterial activity mainly arises from the silver ions held in the interlayer spacing of the titanate. The obtained results should aid the development of more convenient and inexpensive antibacterial implants.