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•KI/γ-Al2O3 catalyst for high yield biodiesel production by oil transesterification.•Modified sol–gel synthesis produces catalyst with superior catalytic performances.•Optimal process ...parameters for transesterification reaction were achieved.•Al-O-K groups take the role as catalytically active centers in transesterification.•Alcoholysis significantly depends on physicochemical properties of catalyst support.
The essence of this study is synthesis of a newly developed and efficient heterogeneous catalyst based on aluminum oxide support promoted with potassium iodide (KI) as active dopant responsible for pronounced catalyst activity. Modified sol–gel, reflux and hydrothermal method were used for synthesis of aluminum-oxide support using aluminum isopropoxide as a precursor. The γ-Al2O3 support and KI/γ-Al2O3 catalyst samples were examined by various experimental techniques. Effects of several operational parameters were considered and those are: methanol to sunflower oil molar ratio, catalyst loadings, reaction time and reaction stirring rate. The obtained results indicated that modified sol–gel method for synthesis of γ-Al2O3 catalyst support is the most favorable for obtaining the catalyst with superior catalytic performances in transesterification of sunflower oil. A maximum conversion (triglycerides to methyl esters) of 99.99% was achieved using the SG-KI/γ-Al2O3-770 catalyst under following optimal process parameters: 15:1 methanol to sunflower oil molar ratio, 2.5 wt% catalysts loading, 600 rpm stirring rate and 4 h of the reaction run.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
In recent years, carbon nanotubes have emerged as a potentially revolutionary material with numerous uses in biomedical applications. Compared to other nanoparticles, discrete multiwalled carbon ...nanotubes (dMWCNTs) have been shown to exhibit advantageous characteristics such as a high surface area-to-volume ratio, biocompatibility, and unique chemical and physical properties. dMWCNTs can be modified to load various molecules such as proteins and nucleic acids and are capable of crossing the cell membrane, making them attractive delivery vehicles for biomolecules. To investigate this, we measured the impact of dMWCNTs on the number of live and dead cells present during different stages of cell proliferation. Furthermore, we used transmission electron microscopy to produce evidence suggesting that dMWCNTs enter the cytoplasm of mammalian cells via an endocytosis-like process and ultimately escape into the cytoplasm. And lastly, we used live-cell staining, qPCR, and a T-cell activation detection assay to quantify the use of dMWCNTs as a delivery vehicle for a toxic, membrane-impermeable peptide, mRNA, siRNA, and a T-cell activating synthetic dsRNA. We demonstrate successful delivery of each payload into a range of cell types, providing further evidence of dMWCNTs as a versatile delivery platform for biomolecular cargo.
Degenerative diseases are a major public health concern for the aging population and mesenchymal stem cells (MSCs) have great potential for treating many of these diseases. However, the quantity and ...quality of MSCs declines with aging, limiting the potential efficacy of autologous MSCs for treating the elderly population.
Human bone marrow (BM)-derived MSCs from young and elderly donors were obtained and characterized using standard cell surface marker criteria (CD73, CD90, CD105) as recommended by the International Society for Cellular Therapy (ISCT). The elderly MSC population was isolated into four subpopulations based on size and stage-specific embryonic antigen-4 (SSEA-4) expression using fluorescence-activated cell sorting (FACS), and subpopulations were compared to the unfractionated young and elderly MSCs using assays that evaluate MSC proliferation, quality, morphology, intracellular reactive oxygen species, β-galactosidase expression, and adenosine triphosphate (ATP) content.
The ISCT-recommended cell surface markers failed to detect any differences between young and elderly MSCs. Here, we report that elderly MSCs were larger in size and displayed substantially higher concentrations of intracellular reactive oxygen species and β-galactosidase expression and lower amounts of ATP and SSEA-4 expression. Based on these findings, cell size and SSEA-4 expression were used to separate the elderly MSCs into four subpopulations by FACS. The original populations (young and elderly MSCs), as well as the four subpopulations, were then characterized before and after culture on tissue culture plastic and BM-derived extracellular matrix (BM-ECM). The small SSEA-4-positive subpopulation representing ~ 8% of the original elderly MSC population exhibited a "youthful" phenotype that was similar to that of young MSCs. The biological activity of this elderly subpopulation was inhibited by senescence-associated factors produced by the unfractionated parent population. After these "youthful" cells were isolated and expanded (three passages) on a "young microenvironment" (i.e., BM-ECM produced by BM cells from young donors), the number of cells increased ≈ 17,000-fold to 3 × 10
cells and retained their "youthful" phenotype.
These results suggest that it is feasible to obtain large numbers of high-quality autologous MSCs from the elderly population and establish personal stem cell banks that will allow serial infusions of "rejuvenated" MSCs for treating age-related diseases.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for ...disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers' response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Salivary gland (SG) dysfunction, due to radiotherapy, disease, or aging, is a clinical manifestation that has the potential to cause severe oral and/or systemic diseases and compromise quality of ...life. Currently, the standard-of-care for this condition remains palliative. A variety of approaches have been employed to restore saliva production, but they have largely failed due to damage to both secretory cells and the extracellular matrix (niche). Transplantation of allogeneic cells from healthy donors has been suggested as a potential solution, but no definitive population of SG stem cells, capable of regenerating the gland, has been identified. Alternatively, mesenchymal stem cells (MSCs) are abundant, well characterized, and during SG development/homeostasis engage in signaling crosstalk with the SG epithelium. Further, the trans-differentiation potential of these cells and their ability to regenerate SG tissues have been demonstrated. However, recent findings suggest that the "immuno-privileged" status of allogeneic adult MSCs may not reflect their status post-transplantation. In contrast, autologous MSCs can be recovered from healthy tissues and do not present a challenge to the recipient's immune system. With recent advances in our ability to expand MSCs in vitro on tissue-specific matrices, autologous MSCs may offer a new therapeutic paradigm for restoration of SG function.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and ...batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions.
Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates.
Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.
Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The study of hydrogen peroxide reduction in an alkaline aqueous medium on a composite of palladium particles dispersed in an amorphous carbon matrix (Pd@AC) was performed. Scanning Electron ...Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FTIR), and X-ray Powder Diffraction (XRD) were used to examine morphology, composition, and crystalline structure. The crystalline structure and size of palladium were analyzed using XRD. The reduction of hydrogen peroxide was studied through cyclic and square-wave voltammetry in an alkaline solution in the absence and presence of dissolved oxygen. The overall electrocatalytic effect of Pd@AC toward hydrogen peroxide reduction strongly depends on the presence of oxygen and the hydrogen peroxide disproportionation reaction in the three-phase system of amorphous carbon, palladium particles, and electrolyte solution.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Current treatments for salivary gland (SG) hypofunction are palliative and do not address the underlying cause or progression of the disease. SG-derived stem cells have the potential to treat SG ...hypofunction, but their isolation is challenging, especially when the tissue has been damaged by disease or irradiation for head and neck cancer. In the current study, we test the hypothesis that multipotent bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model are capable of trans-differentiating to the SG epithelial cell lineage when induced by a native SG-specific extracellular matrix (SG-ECM) and thus may be a viable substitute for repairing damaged SGs.
Rat BM-MSCs were treated with homogenates of decellularized rat SG-ECM for one hour in cell suspension and then cultured in tissue culture plates for 7 days in growth media. By day 7, the cultures contained cell aggregates and a cell monolayer. The cell aggregates were hand-selected under a dissecting microscope, transferred to a new tissue culture dish, and cultured for an additional 7 days in epithelial cell differentiation media. Cell aggregates and cells isolated from the monolayer were evaluated for expression of SG progenitor and epithelial cell specific markers, cell morphology and ultrastructure, and ability to form SG-like organoids in vivo.
The results showed that this approach was very effective and guided the trans-differentiation of a subpopulation of CD133-positive BM-MSCs to the SG epithelial cell lineage. These cells expressed amylase, tight junction proteins (Cldn 3 and 10), and markers for SG acinar (Aqp5 and Mist 1) and ductal (Krt 14) cells at both the transcript and protein levels, produced intracellular secretory granules which were morphologically identical to those found in submandibular gland, and formed SG-like organoids when implanted in the renal capsule in vivo.
The results of this study suggest the feasibility of using autologous BM-MSCs as an abundant source of stem cells for treating SG hypofunction and restoring the production of saliva in these patients.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Oral health plays a significant role in the quality of life and overall well-being of the aging population. However, age-related changes in oral health are not well understood due to challenges with ...current animal models. In this study, we analyzed the oral health and microbiota of a short-lived non-human primate (i.e., marmoset), as a step towards establishing a surrogate for studying the changes that occur in oral health during human aging. We investigated the oral health of marmosets using cadaveric tissues in three different cohorts: young (aged ≤6 years), middle-aged, and older (>10 years) and assessed the gingival bacterial community using analyses of the V3–V4 variable region of 16S rRNA gene. The oldest cohort had a significantly higher number of dental caries, increased dental attrition/erosion, and deeper periodontal pocket depth scores. Oral microbiome analyses showed that older marmosets had a significantly greater abundance of Escherichia-Shigella and Propionibacterium, and a lower abundance of Agrobacterium/Rhizobium at the genus level. Alpha diversity of the microbiome between the three groups showed no significant differences; however, principal coordinate analysis and non-metric multidimensional scaling analysis revealed that samples from middle-aged and older marmosets were more closely clustered than the youngest cohort. In addition, linear discriminant analysis effect size (LEFSe) identified a higher abundance of Esherichia-Shigella as a potential pathogenic biomarker in older animals. Our findings confirm that changes in the oral microbiome are associated with a decline in oral health in aging marmosets. The current study suggests that the marmoset model recapitulates some of the changes in oral health associated with human aging and may provide opportunities for developing new preventive strategies or interventions which target these disease conditions.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Mesenchymal stem cells (MSCs) are highly responsive to cues in the microenvironment (niche) that must be recapitulated ex vivo to study their authentic behavior. In this study, we hypothesized that ...native bone marrow (BM)- and adipose (AD)-derived extracellular matrices (ECM) were unique in their ability to control MSC behavior. To test this, we compared proliferation and differentiation of bone marrow (BM)-derived MSCs when maintained on native decellularized ECM produced by BM versus AD stromal cells (i.e. BM- versus AD-ECM).
We found that both ECMs contained similar types of collagens but differed in the relative abundance of each. Type VI collagen was the most abundant (≈60% of the total collagen present), while type I was the next most abundant at ≈30%. These two types of collagen were found in nearly equal proportions in both ECMs. In contrast, type XII collagen was almost exclusively found in AD-ECM, while types IV and V were only found in BM-ECM. Physically and mechanically, BM-ECM was rougher and stiffer, but less adhesive, than AD-ECM.
During 14 days in culture, both ECMs supported BM-MSC proliferation better than tissue culture plastic (TCP), although MSC-related surface marker expression remained relatively high on all three culture surfaces. BM-MSCs cultured in osteogenic (OS) differentiation media on BM-ECM displayed a significant increase in calcium deposition in the matrix, indicative of osteogenesis, while BM-MSCs cultured on AD-ECM in the presence of adipogenic (AP) differentiation media showed a significant increase in Oil Red O staining, indicative of adipogenesis. Further, culture on BM-ECM significantly increased BM-MSC-responsiveness to rhBMP-2 (an osteogenic inducer), while culture on AD-ECM enhanced responsiveness to rosiglitazone (an adipogenic inducer).
These findings support our hypothesis and indicate that BM- and AD-ECMs retain unique elements, characteristic of their tissue-specific microenvironment (niche), which promote retention of MSC differentiation state (i.e. “stemness”) during expansion and direct cell response to lineage-specific inducers. This study provides a new paradigm for precisely controlling MSC fate to a desired cell lineage for tissue-specific cell-based therapies.
•Tissue culture plastic is an inadequate surface for studying cell behavior in vitro.•Cell-derived ECMs have been shown to recapitulate the stem cell microenvironment.•These ECMs direct MSC response to lineage-specific induction media and factors.•This novel culture system facilitates modeling of physiologic cell behavior in vitro.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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