Effect of Lactoferrin on Odontogenic Differentiation of Stem Cells Derived from Human 3rd Molar Tooth Germ

Stem cell technology has been a great hope for the treatment of many common tissue regeneration-related diseases. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors such as biomaterials or growth factors, in the concept of stem cell conversion into odontogenic cell. Even though lactoferrin has been reported to promote bone growth in vivo, the molecular mechanism of teeth formation has not been elucidated yet. Different concentrations of lactoferrin were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic differentiation of human tooth germ stem cells (hTGSCs) was assessed by gene expression analysis, determination of protein levels in odontogenic differentiation-related protein, measuring alkaline phosphatase (ALP) activity, mineralization, and calcium deposit levels. Lactoferrin-treated group showed the highest ALP activity as opposed to the other groups which were untreated. In addition, the gene expression levels as well as the protein levels of odontogenic factors were found to be high in compared to the control groups. In the current study, it is shown for the first time that there is a significant increase in odontogenic differentiation capacity in hTGSCs when lactoferrin is applied in vitro. The study offers a considerable promise for the development of pulp regeneration by using stem cell technology combined with lactoferrin in functional tooth tissue engineering.     

Neuregulin-1 Increases Connexin-40 and Connexin-45 Expression in Embryonic Stem Cell-Derived Cardiomyocytes

Neuregulin-1 (NRG-1) is a vital factor involved in heart development. NRG-1 up-regulated connexin-40 (Cx40) in mice fetal cardiomyocytes has been reported, while the effect of NRG-1 on expression of connexins in embryonic stem cells-derived cardiomyocytes (ESCMs) is limited studied. The process of cardiomyocytes differentiated from embryonic stem cells with or without NRG-1 treatment was observed continuously. Exposure to NRG-1 increased the expression of Cx40 and connexin-45 (Cx45) in ESCMs, while the expression of connexin-43 was unchanged regardless of NRG-1 treatment Western blot analysis also confirmed that the expression of Cx40 and Cx45 in the beating foci was increased in the presence of NRG-1. These results indicate that connexins are differentially regulated by exogenous NRG-1 during cardiomyocytic differentiation of embryonic stem cells.     

Implications and limitations of cellular reprogramming for psychiatric drug development

Human-induced pluripotent stem cells (hiPSCs) derived from somatic cells of patients have opened possibilities for in vitro modeling of the physiology of neural (and other) cells in psychiatric disease states. Issues in early stages of technology development include (1) establishing a library of cells from adequately phenotyped patients, (2) streamlining laborious, costly hiPSC derivation and characterization, (3) assessing whether mutations or other alterations introduced by reprogramming confound interpretation, (4) developing efficient differentiation strategies to relevant cell types, (5) identifying discernible cellular phenotypes meaningful for cyclic, stress induced or relapsing–remitting diseases, (6) converting phenotypes to screening assays suitable for genome-wide mechanistic studies or large collection compound testing and (7) controlling for variability in relation to disease specificity amidst low sample numbers. Coordination of material for reprogramming from patients well-characterized clinically, genetically and with neuroimaging are beginning, and initial studies have begun to identify cellular phenotypes. Finally, several psychiatric drugs have been found to alter reprogramming efficiency in vitro, suggesting further complexity in applying hiPSCs to psychiatric diseases or that some drugs influence neural differentiation moreso than generally recognized. Despite these challenges, studies utilizing hiPSCs may eventually serve to fill essential niches in the translational pipeline for the discovery of new therapeutics.     

Fatty acid desaturation index in human plasma: comparison of different analytical methodologies for the evaluation of diet effects

Stearoyl-CoA desaturase 1 (SCD1) plays a role in the development of obesity and related conditions, such as insulin resistance, and potentially also in neurological and heart diseases. The activity of SCD1 can be monitored using the desaturation index (DI), the ratio of product (16:1n-7 and 18:1n-9) to precursor (16:0 and 18:0) fatty acids. Here, different analytical strategies were applied to identify the method which best supports SCD1 biology. A novel effective approach was the use of the SCD1-independent fatty acid (16:1n-10) as a negative control. The first approach was based on a simple extraction followed by neutral loss triglyceride fatty acid analysis. The second approach was based on the saponification of triglycerides followed by fatty acid analysis (specific for the position of the double bond within monounsaturated fatty acids (MUFAs)). In addition to the analytical LC-MS assays, different matrices (plasma total triglyceride fraction and the very low-density lipoprotein (VLDL) fraction) were investigated to identify the best for studying changes in SCD1 activity. Samples from volunteers on a high-carbohydrate diet were analyzed. Both ultra HPLC (UHPLC)-MS-based assays showed acceptable accuracies (75–125 % of nominal) and precisions (<20 %) for the analysis of DI-specific fatty acids in VLDL and plasma. The most specific assay for the analysis of the liver SCD activity was then validated for specificity and selectivity, intra- and interday accuracy and precision, matrix effects, dilution effects, and analyte stability. After 3 days of high-carbohydrate diet, only the specific fatty acids in human plasma VLDL showed a significant increase in DI and associated SCD1 activity.     

Isolation and Characterization of Adipose-derived Mesenchymal Stem Cells (ADSCs) from Cattle

Adipose-derived mesenchymal stem cells (ADSCs) were isolated from the adult adipose tissue of 2-year-old cattle, and then characterized by immunofluorescence and RT-PCR. We found that primary bADSCs could be expanded for 25 passages. Expression of β-integrin, CD44, and CD73 was observed by immunofluorescence and RT-PCR. Passage 3 bADSCs were successfully induced to differentiate into osteoblasts and adipocytes. The results indicate the potential for multi-lineage differentiation of bADSCs that may represent an ideal candidate for cellular transplantation therapy.     

Efficient Production of Native Lunasin with Correct N-terminal Processing by Using the pH-Induced Self-Cleavable Ssp DnaB Mini-intein System in Escherichia coli

To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.     

Modified Hyaluronan Hydrogels Support the Maintenance of Mouse Embryonic Stem Cells and Human Induced Pluripotent Stem Cells

These studies provide evidence for the ability of a commercially available, defined, hyaluronan‐gelatin hydrogel, HyStem‐C?, to maintain both mouse embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs) in culture while retaining their growth and pluripotent characteristics. Growth curve and doubling time analysis show that mESCs and hiPSCs grow at similar rates on HyStem‐C? hydrogels and mouse embryonic fibroblasts and Matrigel?, respectively. Immunocytochemistry, flow cytometry, gene expression and karyotyping reveal that both human and murine pluripotent cells retain a high level of pluripotency on the hydrogels after multiple passages. The addition of fibronectin to HyStem‐C? enabled the attachment of hiPSCs in a xeno‐free, fully defined medium.

     

Phase solubility and FTIR-ATR studies of idebenone/sulfobutyl ether β-cyclodextrin inclusion complex

The favourable accessibility offered by sulfobutyl ether β-cyclodextrin (SBE-β-CD) for the complexation with idebenone (IDE) has been probed, as a function of temperature, in liquid state, by phase solubility study, and, in solid state, by FTIR-ATR technique. The phase solubility results indicated the formation of a IDE/SBE-β-CD inclusion complex with 1:1 molar ratio (A_L type diagram), whose apparent stability constants at T = 300, 310, and 320 K have been estimated according to the Higuchi–Connors method. The formation of the inclusion complex has been confirmed on a freeze-dried and a co-precipitated product by FTIR-ATR spectroscopy, monitoring the changes induced by complexation on some characteristic vibrational bands of IDE. Quantitative studies, performed in a wide T range, from T = 250 K to T = 340 K, allowed us to extract information on the effect of temperature on the different hydrogen-bonded environments involving host, guest, and crystallization water molecules. Again, complexation is proved to enhance the stability of the guest, at least in the explored T range.     

Synthesis and preparation of nanoparticles composed of amphiphilic poly(γ-glutamic acid) with different hydrophobic side chains and their potential of membrane disruptive activity

In the development of nanoparticle-based vaccine adjuvants, the interaction between nanoparticles (NPs) and the cells is a key factor. To control them, we focused on the relationship between the hydrophobicity of the side chains and the cell membrane. In this study, amphiphilic poly(γ-glutamic acid) (γ-PGA), using various types of hydrophobic side chains, was synthesized and used to prepare NPs for evaluating the membrane disruptive activity. When leucine ethyl ester (Leu), methionine ethyl ester (Met), or tryptophan ethyl ester (Trp) was grafted, each polymer formed monodispersed NPs at physiological conditions. Significantly, NPs composed of Leu and Trp showed a membrane disruptive activity at the endosomal environment (pH 5–6.5), while NPs composed of Met did not show. This might be due to the weak hydrophobicity of Met compared to that of Leu and Trp, which demonstrated that the interaction between NPs and cells could be controlled by designing the polymer compositions.     

Preparation and properties of insoluble forms of bacteriophage T4 lysozyme and chicken egg white lysozyme

Bacteriophage T4 lysozyme and chicken egg white lysozyme were covalently bound to cyanogen bromide activated Sepharose and to glutaraldehyde activated polyacrylhydrazido-Sepharose. The latter method seemed less favorable for T4 lysozyme, since the poly-acrylhydrazido-agarose conjugate exhibited low activity compared to the agarose conjugate. Whole bacteria (M.luteus and chloroform-treatedE. coli B cells) and the soluble uncross-linked peptidoglycan polymer fromM. luteus were used as substrates. Both types of conjugates exhibited low specific activity (lytic activity) toward insoluble substrates (cells), but surprisingly high specific activity toward the soluble substrate (hydrolytic activity). Product analysis showed that the enzyme conjugates retained their specificity of action, i.e., the same products were formed, and their rates of production were the same as those observed with the soluble (native) enzyme. The cell wall disaccharide-tetrapeptide GlcNAc-MurNAc-L-ala-D-gIu-(A₂pm-D-Ala) (C₆) inhibits the hydrolytic activity of both the native and the agarose bound T4 lysozyme. Only a slightly increased thermal stability was observed upon immobilization of T4 lysozyme, whereas the stability of the enzyme during storage and handling was greatly improved. The pH optimum of the lytic activity of Sepharose-T4 lysozyme was shifted about 1 pH unit to the alkaline side, compared to that found for the soluble enzyme, whereas no pH shift was observed for the polyacrylhydrazido-Sepharose conjugate. The optimum of the hydrolytic activity of Sepharose-T4 lysozyme was shifted to the acidic side. The pH optima of the lytic activity of the various lysozymes toward the bacterial cells were all very similar (>7), and differed greatly from the pH optima (<6) observed for their hydrolytic activities toward the negatively charged soluble peptidoglycan polymer. It is proposed that the observed differences in pH optima primarily reflect the basically different properties measured, i.e., the β(1–4) cleaving activity (hydrolytic activity), and dissolution process of the damaged cells (lytic activity).     

Preparation and characterization of electrospun nanofiber-coated membrane separators for lithium-ion batteries

Nanofiber-coated membrane separators were prepared by electrospinning polyvinylidene fluoride-co-chlorotrifluoroethylene (PVDF-co-CTFE) nanofibers onto three different microporous membrane substrates. The nanofibers on the membrane substrates showed uniform morphology with average fiber diameters ranging from 129 to 134 nm. Electrolyte uptakes, ionic conductivities, and interfacial resistances were studied by soaking the nanofiber-coated membrane separators with a liquid electrolyte solution of 1 M lithium hexafluorophosphate in ethylene carbonate/dimethylcarbonate/ethylmethyl carbonate (1:1:1 by volume). Compared with uncoated membranes, nanofiber-coated membranes had greater electrolyte uptakes and lower interfacial resistances to the lithium electrode. It was also found that after soaking in the liquid electrolyte solution, nanofiber-coated membranes exhibited higher ionic conductivities than uncoated membranes. In addition, lithium-ion half cells containing nanofiber-coated membranes were evaluated with a LiFePO₄ cathode for charge–discharge capacities and cycle performance. The cells containing a nanofiber-coated separator membrane showed high discharge specific capacities and good cycling stability at room temperature. Results demonstrated that coating PVDF-co-CTFE nanofibers onto microporous membrane substrates is a promising approach to obtain new and high-performance separators for rechargeable lithium-ion batteries.     

A wide range optical pH sensor for living cells using Au@Ag nanoparticles functionalized carbon nanotubes based on SERS signals

p-Aminothiophenol (pATP) functionalized multi-walled carbon nanotubes (MWCNTs) have been demonstrated as an efficient pH sensor for living cells. The proposed sensor employs gold/silver core-shell nanoparticles (Au@Ag NPs) functionalized MWCNTs hybrid structure as the surface-enhanced Raman scattering (SERS) substrate and pATP molecules as the SERS reporters, which possess a pH-dependent SERS performance. By using MWCNTs as the substrate to be in a state of aggregation, the pH sensing range could be extended to pH 3.0～14.0, which is much wider than that using unaggregated Au@Ag NPs without MWCNTs. Furthermore, the pH-sensitive performance was well retained in living cells with a low cytotoxicity. The developed SERS-active MWCNTs-based nanocomposite is expected to be an efficient intracellular pH sensor for bio-applications.     

On the determination of HCl+ (A,v′) vibrational populations from HCl+ (A→X) fluorescence spectra: direct comparison between photoionization and penning ionization

Using a beam apparatus, we have measured the HCl⁺ (A,v′→X,v″) fluorescence spectra of HCl⁺ (A,v′) ions formed in HeI (58.4 nm), and NeI (73.6 nm) photoionization and, for the first time, in He (2³ S) Penning ionization under single collision conditions with a wavelength bandwidth around 1 nm. In addition, we have studied Ne (3s ³ P _{2, 0}) Penning ionization of HCl at three different collision energies. The procedure and the problems in extracting HCl⁺ (A,v′) vibrational populations from the data are discussed in some detail. Thedirect comparison of photoionization and Penning ionization data allows definitive conclusions to be drawn on the question whether final state interactions in the Penning reaction change the “nascent” vibrational population (determined by electron spectrometry); for He (2³ S)+HCl, such changes are shown to be absent within the experimental uncertainty (<±10%). For Ne (3s ³ P _{2, 0})+HCl, the HCl⁺ (A,v′=0, 1) populations are also found to be close to those measured by electron spectrometry and essentially independent of collision energy in the range 34–96 meV. From measurements of the fluorescence intensity as a function of HCl density, we have evidence for a fast loss of HCl⁺ (A,v′) ions in collisions with HCl (rate constant around 5·10^?9 cm³s^?1).     

Fabrication of cellulose-based scaffold with microarchitecture using a leaching technique for biomedical applications

Providing a conclusive microenvironment for cell growth, proliferation and differentiation is a major developmental strategy in the tissue engineering and regenerative medicine. This is usually achieved in the laboratory by culturing cells in three-dimensional polymer-based scaffolding materials. Here, we describe the fabrication of a cellulose scaffold for tissue engineering purposes from cellulose fiber using a salt leaching method. The 1-n-allyl-3-methylimidazolium chloride (AmimCl) IL was used as a solvent for cellulose. The leaching methodology used in this study offers the unique advantage of providing effective control of scaffold porosity by simply varying cellulose concentration. Morphologic testing of the scaffolds produced revealed pore sizes of 200–500 μm. In addition, the scaffolds had high water adsorption rates and slow degradation rates. To further investigate the suitability of these scaffolds for tissue engineering applications, biocompatibility was checked using an MTT assay and confirmed by Live/Dead^® viability testing. In addition, scanning electron microscopy and DAPI studies and in vivo experiment demonstrated the ability of cells to attach to scaffold surfaces, and a biocompatibility of matrices with cells, respectively. The authors describe the environmentally friendly fabrication of a novel cellulose-based tissue engineering scaffold.     

Ellagic Acid Normalizes Mitochondrial Outer Membrane Permeabilization and Attenuates Inflammation-Mediated Cell Proliferation in Experimental Liver Cancer

Despite great advances in our understanding of the molecular causes of liver cancer, significant gaps still remain in our knowledge of the disease pathogenesis and development of effective strategies for early diagnosis and treatment. The present study was conducted to evaluate the chemopreventive activity of ellagic acid (EA) against experimental liver cancer in rats. This is the first report that implies a possible role of EA in controlling liver cancer through activation of mitochondrial outer membrane permeability via activating proteins such as Bax, bcl-2, cyt-C, and caspase-9, which play important roles in apoptosis. Downregulation of NF-κB, cyclin D1, cyclin E1, matrix metalloproteinases (MMP)-2, MMP-9, and proliferating cell nuclear antigen (PCNA) were noted in EA-treated experimental rats and controlled inflammation mediated liver cancer when compared to the diethylnitrosamine (DEN)-induced group. Transmission electron microscopy (TEM) analysis of the livers of experimental rats demonstrated that EA treatment renovated its internal architecture. Overall, these results demonstrate the value of molecular approaches in identifying the potential role of EA as an effective chemopreventive agent.     

Biomimetic star-shaped porphyrin-cored poly(l-lactide)-b-glycopolymer block copolymers for targeted photodynamic therapy

Porphyrin-cored poly(l-lactide) (SPPLA) was successfully synthesized from ring-opening polymerization (ROP) of l-lactide initiated with porphyrin core. Then, SPPLA was coupled with benzylsulfanylthiocarbonylsufanylpropionic acid (BSPA), and a macro-reversible addition-fragmentation chain transfer (macroRAFT) polymerization agent SPPLA-BSPA was obtained. Finally, star-shaped porphyrin-cored poly(l-lactide)-b-poly(gluconamidoethyl methacrylate) (SPPLA-b-PGAMA) block copolymers were synthesized via RAFT of unprotected gluconamidoethyl methacrylate (GAMA) in 1-methyl-2-pyrrolidinone (NMP) solution at 70 °C. The structure of this block copolymer was thoroughly studied by nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). Under the irradiation, such SPPLA-b-PGAMA copolymer exhibits efficient singlet oxygen generation and indicates high fluorescence quantum yields. Notably, with UV–vis and dynamic light scattering (DLS) analysis, SPPLA-b-PGAMA showed a very specific recognition with concanavalin A (ConA). Particularly, MTT shows that the cytotoxicity of SPPLA-b-PGAMA against COS-7 cells was very low and, when given a longer irradiation time, more BEL-7402 cancer cells died, which will be investigated in this study.     

Biomolecular Imaging with a C60-SIMS/MALDI Dual Ion Source Hybrid Mass Spectrometer: Instrumentation,Matrix Enhancement,and Single Cell Analysis

We describe a hybrid MALDI/C₆₀-SIMS Q-TOF mass spectrometer and corresponding sample preparation protocols to image intact biomolecules and their fragments in mammalian spinal cord, individual invertebrate neurons, and cultured neuronal networks. A lateral spatial resolution of 10 μm was demonstrated, with further improvement feasible to 1 μm, sufficient to resolve cell outgrowth and interconnections in neuronal networks. The high mass resolution (>13,000 FWHM) and tandem mass spectrometry capability of this hybrid instrument enabled the confident identification of cellular metabolites. Sublimation of a suitable matrix, 2,5-dihydroxybenzoic acid, significantly enhanced the ion signal intensity for intact glycerophospholipid ions from mammalian nervous tissue, facilitating the acquisition of high-quality ion images for low-abundance biomolecules. These results illustrate that the combination of C₆₀-SIMS and MALDI mass spectrometry offers particular benefits for studies that require the imaging of intact biomolecules with high spatial and mass resolution, such as investigations of single cells, subcellular organelles, and communities of cells. Graphical Abstract

?     

Use of hydroxylapatite composite membranes for analysis of bisphenol A

This study evaluates solid-phase micro-extraction (SPME) coupled with gas chromatography–mass spectrometry (GC–MS) to determine trace levels of bis-phenol A in water and leached from plastic containers. In our study, we used very thin composite membranes prepared in the laboratory. The extraction using headspace post-derivatization with bis(trimethylsilyl) trifluoroacetamide (BSTFA), containing 1 % trimethylchlorosilane (TMCS) vapor, following SPME was compared with extraction without derivatization. The SPME experimental procedures to extract bis-phenol A in water were optimized with a relatively polar polyacrylate (PA)-coated fiber, an extraction time of 50 min, and desorption at 300 °C for 2 min. Headspace derivatization following SPME was performed using 7 μL of BSTFA with 1 % TMCS at 65 °C for 30 s. The precision was 5.2 % without derivatization and 9.0 % headspace derivatization. The detection limit was determined to be at the nanogram per liter level. When SPME was used following headspace derivatization, the detection limit was one order of magnitude better than that achieved without derivatization. The results of this study reveal the adequacy of the SPME–GC–MS method for analyzing bisphenol A leached from plastic containers. The concentrations of bisphenol A leached from plastic containers into water ranged from 0.7 to 78.5 μg L^?1.