Adipose-derived stem cells-conditioned medium improved osteogenic differentiation of induced pluripotent stem cells when grown on polycaprolactone nanofibers

Considering that the common osteogenic growth factors cannot be transplanted with stem cells to the patients, many studies are underway to find a replacement for these factors. Recently, it has been determined that mesenchymal stem cell (MSC)-derived conditioned medium (CM) contains effective factors in the bone formation process. In the current study, the synergistic effect of adipose-derived MSC’s CM, and polycaprolactone (PCL) scaffold was investigated on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). After scaffold fabrication by electrospinning and characterization by scanning electron microscopy, iPSCs proliferation in the presence of CM, PCL, and both was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide. Then, iPSCs osteogenic differentiation was investigated while cultured on tissue culture plate and PCL under CM compared with the osteogenic medium using alizarin red staining, calcium content, alkaline phosphatase activity and gene and protein expression analysis. Proliferation rate of the iPSCs was increased while cultured under CM and its effect was synergistically enhanced by culture on PCL. Evaluation of the osteogenic markers was showed CM alone could induce osteogenic differentiation into the iPSCs and this potential was significantly increased while combined with PCL nanofibrous scaffold. According to the results, it was demonstrated that CM has an osteogenic induction property almost the same of the common osteogenic medium and it can also be used potentially with stem cells when transplant to the patients. CM can also help by prolonging cell survival at the site of the defect as well as accelerating healing process.     

Acidic residue modifications restore chaperone activity of β-casein interacting with lysozyme

An important factor in medicine and related industries is the use of chaperones to reduce protein aggregation. Here we show that chaperone ability is induced in β-casein by modification of its acidic residues using Woodward's Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study β-casein chaperone activities. The mechanism for chaperone activity of the modified β-casein was determined using UV-vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoretical calculations. Our results indicated that the β-casein destabilizes the lysozyme and increases its aggregation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of β-casein resulting in its altered net negative charge upon interactions with lysozyme. The reversible stability of lysozyme increased in the presence of WRK-modified β-casein, and hence its aggregation rate decreased. These results demonstrate the enhanced chaperone activity of modified β-casein and its protective effects on lysozyme refolding.     

Effects of Influenza Derived Peptide on CD8 T Cell Responses to MHC Class I-Restricted Human Telomerase Reverse Transcriptase (hTERT)-Derived Peptide

International Journal of Peptide Research and Therapeutics - Tumor cells in breast cancer are immunogenic and express proteins that can induce immune responses. One important antigen is human...     

Construction and immunogenicity of a new Fc-based subunit vaccine candidate against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

As an ancient disease, tuberculosis (TB) is a major global health threat. Therefore, there is an urgent need for an effective and safe anti-TB vaccine. In the current study, a delivery system of Fc domain of mouse IgG2a and early secreted antigenic target protein 6 (ESAT-6) was evaluated for the selective uptake of antigens by antigen-presenting cells (APCs). Thus, it was based on the immunogenicity of a fusion protein. The study was initiated by the transfer of recombinant expression vectors of pPICZαA-ESAT-6:Fcγ2a and pPICZαA-ESAT-6: His into Pichia pastoris (P. pastoris). Recombinant proteins were assessed for immunogenicity following the immunoblotting analysis. High levels of IFN-γ and IL-12 were produced to induce Th1-type cellular responses through vaccination with both recombinant proteins [ESAT-6:Fcγ2a (EF) and ESAT-6:His (EH)]. The Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low increment in IL-4 compared to PBS, BCG, and EH groups. Although in all the immunized groups, the ratio of IFN-γ/IL-4 was in favor of Th1 responses, the highest Th1/Th2 balance was observed in EF immunized group. Fc fragment of mouse IgG2a may induce a selective uptake of APCs towards the cross-presentation and formation of Th1 responses in favor of an appropriate protective anti-tuberculosis reaction. Thus, further research on Fc-fusion proteins is required to develop Fc-based TB vaccines.     

The polydispersity of αB-crystallin is rationalized by an interconverting polyhedral architecture

We report structural models for the most abundant oligomers populated by the polydisperse molecular chaperone αB-crystallin. Subunit connectivity is determined by using restraints obtained from nuclear magnetic resonance spectroscopy and mass spectrometry measurements, enabling the construction of various oligomeric models. These candidate structures are filtered according to their correspondence with ion-mobility spectrometry data and cross-validated by using electron microscopy. The ensuing best-fit structures reveal the polyhedral architecture of αB-crystallin oligomers, and provide a rationale for their polydispersity and facile interconversion.     

Convergent Functional Genomics of Oligodendrocyte Differentiation Identifies Multiple Autoinhibitory Signaling Circuits

Inadequate remyelination of brain white matter lesions has been associated with a failure of oligodendrocyte precursors to differentiate into mature, myelin-producing cells. In order to better understand which genes play a critical role in oligodendrocyte differentiation, we performed time-dependent, genome-wide gene expression studies of mouse Oli-neu cells as they differentiate into process-forming and myelin basic protein-producing cells, following treatment with three different agents. Our data indicate that different inducers activate distinct pathways that ultimately converge into the completely differentiated state, where regulated gene sets overlap maximally. In order to also gain insight into the functional role of genes that are regulated in this process, we silenced 88 of these genes using small interfering RNA and identified multiple repressors of spontaneous differentiation of Oli-neu, most of which were confirmed in rat primary oligodendrocyte precursors cells. Among these repressors were CNP, a well-known myelin constituent, and three phosphatases, each known to negatively control mitogen-activated protein kinase cascades. We show that a novel inhibitor for one of the identified genes, dual-specificity phosphatase DUSP10/MKP5, was also capable of inducing oligodendrocyte differentiation in primary oligodendrocyte precursors. Oligodendrocytic differentiation feedback loops may therefore yield pharmacological targets to treat disease related to dysfunctional myelin deposition.Demyelination and/or incomplete remyelination, followed by axonal loss and neuronal death, is associated with several neurodegenerative disorders, including multiple sclerosis (37), Pelizaeus-Merzbacher disease, and spastic paraplegia type 2 (16), while myelin abnormalities are also seen for psychiatric disorders, including major depression (2), schizophrenia, and autism (reviewed in reference 10). In humans, the central neural system consists of an unusually high proportion (50%) of white matter, which is normally maintained by proliferating, migrating, and remyelinating oligodendrocytes (10). However, in, for instance, secondary progressive multiple sclerosis, at a stage where the autoimmune insult has abated, myelin degeneration continues. This insufficiency of myelin repair has been attributed to a failure of oligodendrocyte precursors to differentiate (11, 20).In order to better understand which genes play a role in multiple sclerosis, a number of proteomic (13), genomic (25, 29), and genetic (35) approaches have been utilized. However, diseased lesions consist of different admixtures (e.g., contain immune and glia infiltrates) of cell types compared to controls, so such postmortem samples may present differential cell, rather than gene or protein, expression. Therefore, understanding how such genes fit into pathophysiological processes (and in which) is problematic. From population genetic surveys thus far, only a few genes have been found to be associated with multiple sclerosis (31).Based on these considerations, we chose to examine genome-wide gene expression changes in a murine oligodendroglial precursor cell line, Oli-neu (18), as these cells underwent differentiation into myelin basic protein (MBP)-producing cells. Even for a pure cell line, this type of transition is typically associated with changes in the expression of thousands of genes, by itself providing little meaningful information. We therefore decided to look at oligodendrocyte differentiation as induced by different agents, in the hope that from this combined data set one might extract “core genes” whose modulation is closely linked to the differentiation process. The enriched set of genes was further evaluated for their functional involvement in the differentiation process.     

Semi-rational chemical modification of endoinulinase by pyridoxal 5′-phosphate and ascorbic acid

It is important to improve the quality of the enzyme inulinase used in industrial applications without allowing the treatment to have any adverse effects on enzyme activity. We achieved preferential chemical modification of the non-catalytic domain of endoinulinase (EC 3.2.1.7) to enhance the thermostability of the enzyme. We used pyridoxal 5′-phosphate (PLP) to modify the more accessible lysine residues at the surface of endoinulinase and then performed a necessary step of reduction with ascorbate. Endoinulinase was incubated in the presence of PLP at various concentrations; this step was followed by reduction of the resulting Schiff base and dialysis. The effects of different PLP concentrations and incubation times on enzyme modification were evaluated. Enzyme deactivation was observed immediately after treatment, even at low PLP concentrations, while reactivation was observed for samples treated with low PLP concentrations after a period of time. Structural analysis revealed that the α-helix content increased from 13.60% to 17.60% after applying the modification strategy; consequently, enzyme stabilization was achieved. The melting temperature (T_m) of the modified enzyme increased from 64.1 °C to 72.2 °C, and a comparative study of thermal stability at 25 °C, 45 °C, and 50 °C for 150 min confirmed that the enzyme was stabilized because of increase in its half-life (t_1/2) after PLP modification/ascorbate reduction. The modification process was optimized to achieve the optimum mole ratio for the PLP/endoinulinase (1.37). Excess moles of the modifier are thought to be responsible for enzyme deactivation through unwanted/nonspecific and noncovalent interactions, and the optimization ensured that there was no excess modifier after the desired covalent reaction was complete.     

Thermal inactivation and conformational lock of bovine carbonic anhydrase

The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.