Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin,sodium fluoride,melanocyte-stimulating hormone

Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.     

Adipose tissue‐derived mesenchymal stem cells and keratinocytes co‐culture on gelatin/chitosan/β‐glycerol phosphate nanoscaffold in skin regeneration

Using cell‐based engineered skin is an emerging strategy for treating difficult‐to‐heal wounds. To date, much endeavor has been devoted to the fabrication of appropriate scaffolds with suitable biomechanical properties to support cell viability and growth in the microenvironment of a wound. The aim of this research was to assess the impact of adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) and keratinocytes on gelatin/chitosan/β‐glycerol phosphate (GCGP) nanoscaffold in full‐thickness excisional skin wound healing of rats. For this purpose, AD‐MSCs and keratinocytes were isolated from rats and GCGP nanoscaffolds were electrospun. Through an in vivo study, the percentage of wound closure was assessed on days 7, 14, and 21 after wound induction. Samples were taken from the wound sites in order to evaluate the density of collagen fibers and vessels at 7 and 14 days. Moreover, sampling was done on days 7 and 14 from wound sites to assess the density of collagen fibers and vessels. The wound closure rate was significantly increased in the keratinocytes‐AD‐MSCs‐scaffold (KMS) group compared with other groups. The expressions of vascular endothelial growth factor, collagen type 1, and CD34 were also significantly higher in the KMS group compared with the other groups. These results suggest that the combination of AD‐MSCs and keratinocytes seeded onto GCGP nanoscaffold provides a promising treatment for wound healing.     

CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.     

eRepo-ORP: Exploring the Opportunity Space to Combat Orphan Diseases with Existing Drugs

About 7000 rare, or orphan, diseases affect more than 350 million people worldwide. Although these conditions collectively pose significant health care problems, drug companies seldom develop drugs for orphan diseases due to extremely limited individual markets. Consequently, developing new treatments for often life-threatening orphan diseases is primarily contingent on financial incentives from governments, special research grants, and private philanthropy. Computer-aided drug repositioning is a cheaper and faster alternative to traditional drug discovery offering a promising venue for orphan drug research. Here, we present eRepo-ORP, a comprehensive resource constructed by a large-scale repositioning of existing drugs to orphan diseases with a collection of structural bioinformatics tools, including eThread, eFindSite, and eMatchSite. Specifically, a systematic exploration of 320,856 possible links between known drugs in DrugBank and orphan proteins obtained from Orphanet reveals as many as 18,145 candidates for repurposing. In order to illustrate how potential therapeutics for rare diseases can be identified with eRepo-ORP, we discuss the repositioning of a kinase inhibitor for Ras-associated autoimmune leukoproliferative disease. The eRepo-ORP data set is available through the Open Science Framework at https://osf.io/qdjup/.     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

Characterization of two independent modes of action of ATP on human erythrocyte sugar transport

Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP-mediated inhibition of DNA replication and S phase progression: involvement of Rb, p21Cip1, and PCNA

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1 to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1 proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/- and p21Cip1-/- 3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase-specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase-specific antitumor agents.     

Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.     

Comparison of iron-molybdenum cofactor-deficient nitrogenase MoFe proteins by X-ray absorption spectroscopy: implications for P-cluster biosynthesis

Nitrogenase, the enzyme system responsible for biological nitrogen fixation, is believed to utilize two unique metalloclusters in catalysis. There is considerable interest in understanding how these metalloclusters are assembled in vivo. It has been presumed that immature iron-molybdenum cofactor-deficient nitrogenase MoFe proteins contain the P-cluster, although no biosynthetic pathway for the assembly of this complex cluster has been identified as yet. Through the comparison by iron K-edge x-ray absorption edge and extended fine structure analyses of cofactor-deficient MoFe proteins resulting from nifH and nifB deletion strains of Azotobacter vinelandii, a novel [Fe-S] cluster is identified in the DeltanifH MoFe protein. The iron-iron scattering displayed by the DeltanifH MoFe protein is more similar to that of a standard [Fe(4)S(4)]-containing protein than that of the DeltanifB MoFe protein, which is shown to contain a "normal" P-cluster. The iron-sulfur scattering of the DeltanifH MoFe protein, however, indicates differences in its cluster from an [Fe(4)S(4)](Cys)(4) site that may be consistent with the presence of either oxygenic or nitrogenic ligation. Based on these results, models for the [Fe-S] center in the DeltanifH MoFe protein are constructed, the most likely of which consist of two separate [Fe(4)S(4)] sites, each with some non-cysteinyl coordination. This type of model suggests that the P-cluster is formed by the condensation of two [Fe(4)S(4)] fragments, possibly concomitant with Fe protein (NifH)-induced conformational change.