DMEM Medium Research Articles

Boric acid alleviates periodontal inflammation induced by IL-1β in human gingival fibroblasts

BackgroundBoric acid (BA) has been found to have therapeutic effects on periodontal disease through beneficially affecting antibacterial, anti-viral, and anti-inflammatory actions. MethodsThis study was conducted to determine the effect of BA on cell viability and on mRNA expressions of proinflammatory and anti-inflammatory cytokines and on oxidative stress enzymes induced by IL-1β (1 ng/mL) in Human Gingival Fibroblasts (HGF) cultured for 24 and 72 h in DMEM media. The BA concentrations added to the media were 0.09 %, 0.18 %, 0.37 %, and 0.75 %. ResultsAll of the BA concentrations increased the viability of cell cultured in DMEM media only, indicating that these concentrations were not toxic and actually beneficial to cell viability. The addition of 1 ng/m: of IL-1β decreased cell viability that was overcome by all concentrations of BA at both 24 and 72 h. The IL-1β addition to the media increased the expressions of the proinflammatory cytokines IL-1β, IL-6, IL-8, and IL-17; the anti-inflammatory cytokine IL-10; and the oxidative stress enzymes superoxide dismutase (SOD0 and glutathione peroxidase (GPX). The IL-1β induced increase mRNA expression of IL-1β was decreased at 24 h by the 0.37 % and 0.75 % BA additions to the media and decreased in a dose-dependent manner by all concentrations of BA at 72 h. The IL-1β induced increase in the expression of IL-6 was decreased in dose-dependent manner at 72 h by BA. All BA concentrations decreased the IL-1β induced expression of IL-8 at both 24 and 72 h. The induced increase in IL-17 by IL-1β was not significantly affected by the BA additions. The increase in the anti-inflammatory cytokine IL10 induced by IL-1β was increased further by all BA additions in dose dependent manner at both 24 and 72 h. The mRNA expressions of SOD and GPX increased by IL-1β were further increased by the 0.37 % and 0.75 % BA concentrations at 72 h. ConclusionsThese findings indicate that BA can significantly modulate the cytokines that are involved in inflammatory stress and reactive oxygen species action and thus could be an effective therapeutic agent in the treatment of periodontal disease.

Influence of Different Glucose Concentrations on the Expression of miR-29c-3p microRNA in Mesenchymal Stem Cells.

Background:miR-29c-3pmanages a set of genes involved in regenerative medicine, and It seems that hyperglycemia in diabetic patients influences the power of stem cells to tissue regeneration the difficulties of diabetes by affecting the expressionmiR-29c-3pin mesenchymal stem cells. The study aims to analyze the effect of various glucose concentrations on the miR-29c-3p expressionin mesenchymal stem cells. Materials and Methods:After receiving donated mesenchymal stem cells from Tarbiat Modares University, these cells were cultivated in a DMEM culture medium, including three different concentrations of glucose 250, 140, and 100 mg/dl. RNA was extracted from these cells after 72 hours, the Real-Time PCR technique assessed the expression of miR-29c-3p, and the results were analyzed by REST software. Results:miR-29c-3pexpression in cells at concentrations of 140 and 250 mg/dL compared to typical situations (100 mg/dl) was significantly decreased (P˂0.05), which declined at a concentration of 250 mg/dl was more. Conclusion:ReducedmiR-29c-3pexpression in mesenchymal stem cells in chronic and mild diabetic situations demonstrated that diabetes might be one of the significant reasons for mesenchymal stem cells' reduced ability to repair tissue damage.

New parameters for in vitro development of cell lines of the species Astyanax bimaculatus (Linnaeus, 1758) and Geophagus proximus (Castelnau, 1855).

Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz-L-15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L-15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures.

WITHDRAWN: Transdifferentiation of Human Neonatal Foreskin Fibroblasts into Keratinocyte-like Cells via Regulating the AKT/P53/WNT/LEF1 Axis

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Novel Synthesis of Fluorescein Isothiocyanate-Based Fluorescent Nanoprobes in Imaging Lung Inflammation

We aimed to examine the novel synthesis of fluorescent nanoprobes synthesized in imaging lung inflammation and diseased tissues. All reagents were purchased from commercial suppliers to synthesize the PLGA, PEG, GFP, RFP, rhodamine, and magnetic fluorescent nanoprobes. We performed experiments using human lung cells from the Chinese Academy of Medical Sciences Cell Center. The cells were cultured in a DMEM medium. Confocal microscopy was used to label the cells during imaging. All statistical analyses were performed in GraphPad Prism. There were significant differences in the fluorescent intensities of all nanoprobes. The fluorescence intensity of the iron oxide nanoprobes was significantly higher than all other probes, while Cy5.5 and RFP nanoprobes had significantly higher fluorescence intensity than PEG and FITC. Moreover, we found that GFP has a better quantum yield than RFP, while RFP has longer emission wavelengths than GFP. Fluorescent nanoprobes have shown great potential as a non-invasive and sensitive tool for imaging lung inflammation and diseased tissue.

Crocus Officinalis (L.) Extract on Human Colerectal Cancer Cell Line (HT-22): Investigation in Vitro

Crocus sativus L. has been used as spice, food colouring and medicinal plant for thousands of years. In this study, antioxidant properties and cytotoxic effect of saffron extract were evaluated in HT-22 cell lines. Malignant and non-malignant cells (L929) were cultured in DMEM medium and incubated with different concentrations of saffron extract in different solvents (water, ethanol, ethyl-acetate, n-hexane). Cell viability was determined by MTT assay. Antioxidant parameters such as DPPH, FRAP, total polyphenol and flavonoid amounts were measured. The highest DPPH value was found as 15.90±0.12 µg/mL in ethyl acetate solvent. Total polyphenols were 89.06±0.02 mg GAE/g extract, flavonoids were 76.98±2.08 µg QE/g extract and FRAP was 67.93±0.98 mg/g extract in ethanol solvent. In colon cancer, IC50 value 94±1.37 µg/mL was found to be the highest value. This is thought to be due to the high antioxidant capacity in ethanol solvent. While antioxidant capacity was not found to be high, antiproliferation effect was found to be high. Saffron may also be considered as a promising chemotherapeutic agent in cancer treatment in the future.

Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

Abstract 2123: Validation of novel histone deacetylases (HDAC) specific nanoparticles using HDACs overexpressed human embryonic kidney cells

Abstract Introduction: Histone deacetylase (HDAC) is a major enzyme system that is upregulated in numerous disease conditions including cancer. Harnessing the upregulation of HDAC can be a viable tool to design targeted therapy for these diseases. Nanoparticle technology has emerged as a powerful workhorse to suppress the survival of diseased cells selectively. As such, we aim to design a nanoscale, drug delivery platform that responds to HDAC enzymes to initiate drug release. In this poster, we show the pharmacological evidence that HDAC-sensitive nanoparticles are interacting with HDAC enzymes in vitro in time and concentration dependent pattern. Methods: We synthesized HDAC-responsive nanoparticles via developing a block copolymer composed of a poly (ethylene glycol) block and a poly (L-acetylated lysine) block. Abbreviated as PEG-acPLK, these copolymers self-assemble as nanoparticles with negatively charged surface. Using in vitro culture of HEK 293 cells, we showed that the formed nanoparticles interact and interfere with HDAC enzymes. Cells were grown to 80-90% confluency in DMEM media w/10% FBS prior to experiments. HEK-293 cells were overexpressed with HDAC 7 & HDAC 8 using Lentiviral vectors. Western blot was done to prove the successful overexpression of the HEK cells. Cytotoxicity assay was done by MTT to analyze the efficacy of HDAC specific nanoparticle on control and overexpressed HEK-293 cells. Results: Our work showed that, PEG-acPLK systems form nanoparticles with a hydrodynamic diameter (RH) of 120-150 nm. The particles were negatively charged on their surface, as such did not produce any non-specific toxicity to cells, such as damage of cell membranes. HEK-293 cells were infected with lentivirus vectors for HDAC 7 and HDAC 8. The medium was changed 8 h after infection. Forty-eight hours after infection, the positive cells were selected with 5 µg/mL puromycin. The lentivirus stable transformants of HEK-293 cells were collected and significant overexpression were confirmed by western blot. For the cytotoxicity study, control and overexpressed cells were treated with HDAC-nanoparticle and PEG-acPLKC (5 µg/mL to 70 µg/mL) for 24hrs. Both the HDAC-nanoparticle and PEG-acPLC (free form) showed more than 60% of toxicity at the concentration of 20 µg/mL. Whereas the HDAC 7 and 8 overexpressed HEK-293 cells did not showed any effects with nanoparticle and Ae-peg-PLKC, suggesting compensating effect due to highly available HDAC system in place and confirm the effectiveness of the HDAC nanoparticle. Summary and Conclusion: Overall, these results showed that HDAC specific nanoparticle exhibited effects in HEK-293 HDACs overexpressed cells. Further molecular mechanistic studies will help to delineate how the HDAC-responsive particles can be used for drug delivery. Citation Format: Sahil Lohana, Yogaraj Ramakrishnan, Premanand Balraj, Md Rakib Hasan Khan, Mallik Sanku, Venkatachalem Sathish, Mohiuddin Quadir. Validation of novel histone deacetylases (HDAC) specific nanoparticles using HDACs overexpressed human embryonic kidney cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2123.

Alcohol extract of Rubia yunnanensis: Metabolic alterations and preventive effects against OGD/R‑induced oxidative damage in HT22 cells.

The present study investigated the inhibitory and neuroprotective effects of Rubia yunnanensis alcohol extract (RY-A) on oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells. In vitro cultured HT22 cells were randomly divided into control, OGD/R, OGD/R + 100 µmol/l edaravone and OGD/R + 10, 20 and 40 µg/ml RY-A groups. Oxygen-sugar deprivation was performed with 10 mmol/l sodium dithionite combined with sugar-free DMEM medium for 2 h, followed by re-glycolization and reoxygenation for 2 h to establish an in vitro OGD/R model. Cell morphology was observed under a phase contrast microscope. Cell survival rate was detected by thiazolyl blue and lactate dehydrogenase and oxidative stress-related indexes were detected by commercial kits. The effects and metabolic alterations of RY-A treatment after OGD/R were evaluated using ultra-high performance liquid chromatography and mass spectrometry. Protein levels were further examined by western blotting. The results showed that cells in the OGD/R group were swollen and lacked protrusions, had significantly reduced viability and had significantly elevated oxidative stress-related indexes of reactive oxygen species, nitric oxide levels and malondialdehyde content and significantly reduced activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, compared with controls. Compared with the OGD/R group, the RY-A group had significantly improved cell morphology and significantly increased cell viability and in terms of oxidative stress, exhibited significantly reduced reactive oxygen species, nitric oxide levels and malondialdehyde content, as well as significantly increased superoxide dismutase and glutathione peroxidase activities. Metabolomic analysis identified changes in 20 metabolites, including L-tryptophan, ornithine, eicosapentaenoic acid-d5, isosafrole and xanthine. Metabolomics analysis showed that the pathways affected included those related to phenylalanine, tyrosine and tryptophan biosynthesis, the prolactin signaling pathway and amphetamine addiction. These results suggested that RY-A had significant preventive effects on an in vitro model of cerebral ischemia-reperfusion injury simulated by OGD/R and the mechanism may be related to increased tryptophan content, activation of indoleamine 2,3-dioxygenase enzymes and inhibition of oxidative stress.

Antimicrobial, anti-adhesive, and anti-invasive effects of condition media derived from adipose mesenchymal stem cells against Shigella flexneri.

The objective of the current study was to examine the antimicrobial, anti-adhesion, and anti-invasion properties of various concentrations of condition media obtained from adipose mesenchymal stem cells (AD-MSCs CM) against Shigella flexneri (S. flexneri). AD-MSCs characterization and antimicrobial assay were performed using flow cytometry and microdilution by colony counting, respectively. For evaluating adhesion and invasion, Caco-2 cells were infected by S. flexneri at three different multiplicities of infection (MOIs of 1, 10, and 50) and then treated with DMEM medium and AD-MSCs CM. The inhibitory effect of AD-MSCs CM was assessed after 24 and 48 h of treatment by CFU (colony-forming unit) counting. A total of 84, 65, and 56% reduction in the adhesion rate of S. flexneri to Caco-2 cells treated with AD-MSCs CM were observed at MOIs of 1, 10, and 50, respectively. While S. flexneri at MOI:1 had no invasive effect on Caco-2 cells, convincing invasion was detected at MOIs of 10 and 50, showing a significant decrease following treatment with AD-MSCs CM. The current study results open new insights into AD-MSCs CM as a new non-antibiotic therapeutic candidate for S. flexneri infections.

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures

Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism.

YTHDF2 as a Mediator in BDNF-Induced Proliferation of Porcine Follicular Granulosa Cells.

In female mammals, the proliferation and apoptosis of granulosa cells (GCs) are critical in determining the fate of follicles and are influenced by various factors, including brain-derived neurotrophic factor (BDNF). Previous research has shown that BDNF primarily regulates GC proliferation through the PI3K/AKT, NF-kB, and CREB tumour pathways; however, the role of other molecular mechanisms in mediating BDNF-induced GC proliferation remains unclear. In this study, we investigated the involvement of the m6A reader YTH domain-containing family member 2 (YTHDF2) in BDNF-stimulated GC proliferation and its underlying mechanism. GCs were cultured in DMEM medium supplemented with varying BDNF concentrations (0, 10, 30, 75, and 150 ng/mL) for 24 h. The viability, number, and cell cycle of GCs were assessed using the CCK-8 assay, cell counting, and flow cytometry, respectively. Further exploration into YTHDF2's role in BDNF-stimulated GC proliferation was conducted using RT-qPCR, Western blotting, and sequencing. Our findings indicate that YTHDF2 mediates the effect of BDNF on GC proliferation. Additionally, this study suggests for the first time that BDNF promotes YTHDF2 expression by increasing the phosphorylation level of the ERK1/2 signalling pathway. This study offers a new perspective and foundation for further elucidating the mechanism by which BDNF regulates GC proliferation.

Identification of Structures Labeled by Indocyanine Green in the Rat Choroid and Retina Can Guide Interpretation of Indocyanine Green Angiography.

Indocyanine green (ICG) is an albumin and lipoprotein binding dye absorbing in the far red used in angiography to visualize choroidal vessels (ICG angiography [ICGA]). To guide interpretation, ICG transport in the choroid, RPE, and retina of rats was studied. Two conditions were used: RPE/choroid organoculture, incubated for 45 minutes in DMEM medium, 1% fetal bovine serum containing 0.25 mg/mL ICG and RPE/choroid and neural retina flat-mounts at 1 and 6 hours after intravenous ICG injection. Early and late sequences of ICGA were recorded until 6 hours. Ultra-deep red confocal microscope was used to localize ICG in flat-mounts and immunohistochemistry was performed for caveolin-1, tryptase (mast cell marker), and tubulin β3 (a nerve marker). In the organoculture, ICG penetrated homogeneously in the cytoplasm and stained the membranes of the RPE. At 1 hour after intravenous injection, ICG appeared in fine granules in RPE, partly labeled with caveolin-1 and decreasing at 6 hours. At 1 hour and 6 hours, ICG was found in the retinal vessels, faintly in the inner retina, and in the photoreceptor outer segments at 6 hours. In the choroid, ICG colocalized with mast cells, immunostained with tryptase, and accumulated along the large tubulin β3-labeled nerve bundles. The hypothesis was raised on the interpretation of late ICGA infrared photography in case of transthyretin amyloidosis with neuropathy. Beside being a vascular dye, ICG is transported from the vessels to the RPE toward the outer retina. It stains mast cells and large choroidal nerves. These observations could help the analysis of ICGA images.

Effects of phosphorus and fenofibrate on lipid deposition, mitochondrial function and protein synthesis in obscure puffer (Takifugu obscurus)

This study aimed to investigate the effects of phosphorus (P) restriction and fenofibrate (F) on lipid deposition, mitochondrial function and protein synthesis in obscure puffer using in vivo and in vitro studies. In vivo, healthy fish with an initial body weight of 18.21 ± 0.04 g were fed three diets supplemented with different P and fenofibrate contents for 10 weeks: Control (5.3 g/kg P), P0 (0 g/kg P) and P0 + F (0 g/kg P and 3.5 g/kg F). The results indicated that P0 treatment significantly reduced weight gain rate and protein deposition, but increased body lipid accumulation, hepatic lipid droplets and serum triglyceride and cholesterol content (P < 0.05). However, P0 + F treatment reversed these. Furthermore, P0 treatment was found to inhibit the mRNA levels of pparα, lpl and cyp7a1, whereas P0 + F treatment significantly increased the expression levels of these genes. In vitro, primary hepatocytes were cultured for 48 h in three different culture media: Control (normal DMEM medium), P0 (DMEM without P), and P0 + F (DMEM without P + 200 mM F). The results showed that P0 significantly decreased the mRNA and protein expression levels of Pparα (P < 0.05), and P0 + F up-regulated mRNA expression levels of pparα compared with P0 group (P < 0.05). Furthermore, P0 inhibited the expression levels of genes associated with fatty acid β-oxidation (cpt1 and acox1) and transport (cd36), but elevated the expression levels of genes related to lipid synthesis (fas, me, g6pd and dgat1) and cholesterol synthesis (hmgcr) (P < 0.05). However, P0 + F reversed these effects. P0 decreased mitochondrial abundance, membrane potential and ATP content, increased the mRNA expression level of ampk and 4epb1 and inhibited the mRNA expression of pgc1-α and the protein expression of mTOR and S6k (P < 0.05), whereas P0 + F increased mitochondrial abundance and membrane potential, increased mtor mRNA expression and inhibited 4ebp1 mRNA expression. Overall, this study suggests that P deficiency leads to lipid and cholesterol accumulation and impaired growth in fish, and fenofibrate can alleviate these effects by improving lipid and cholesterol metabolism, mitochondrial function, and protein synthesis.

Interactions Between Silver Nanoparticles and Culture Medium Biomolecules with Dose and Time Dependencies.

The interaction between silver nanoparticles (AgNPs) and molecules producing coronas plays a key role in cytotoxicity mechanisms. Once adsorbed coronas determine the destiny of nanomaterials in vivo, their effective deployment in the biomedical field requires a comprehensive understanding of the dynamic interactions of biomolecules with nanoparticles. In this work, we characterized 40nm AgNPs in three different nutritional cell media at different molar concentrations and incubation times to study the binding mechanism of molecules on surface nanoparticles. In addition, their cytotoxic effects have been studied in three cell lineages used as tissue regeneration models: FN1, HUV-EC-C, RAW 264.7. According to the data, when biomolecules from DMEM medium were in contact with AgNPs, agglomeration and precipitation occurred. However, FBS medium proteins indicated the formation of coronas over the nanoparticles. Nonetheless, little adsorption of molecules around the nanoparticles was observed when compared to DMEM supplemented with 10% FBS. These findings indicate that when nanoparticles and bioproteins from supplemented media interact, inorganic salts from DMEM contribute to produce large bio-coronas, the size of which varies with the concentration and time. The static quenching mechanism was shown to be responsible for the fluorescence quenching of the bioprotein aggregates on the AgNPs surface. The calculated bioprotein-nanoparticle surface binding constants were on the order of 105M-1 at 37°C, with hydrophobic interactions driven by enthalpy and entropy playing a role, as confirmed by thermodynamic analysis. Cytotoxicity data showed a systematic degrowth in the viable cell population as the number of nanoparticles increased and the diameter of coronas decreased. Cytotoxic intervals associated with half decrease of cell population were established for AgNPs molar concentration of 75µM for 24h and 50µM for 48h. In summary, through the cytotoxicity mechanism of bio-coronas we are able to manipulate cells' expansion rates to promote specific processes, such inflammatory mechanisms, at different time instants.

Comparison of the differentiation of ovine fetal bone-marrow mesenchymal stem cells towards osteocytes on chitosan/alginate/CuO-NPs and chitosan/alginate/FeO-NPs scaffolds

In the current study, the creation of a chitosan/alginate scaffold hydrogel with and without FeO-NPs or CuO-NPs was studied. From fetal ovine bone marrow mesenchymal stem cells (BM-MSCs) were isolated and cultivated. Their differentiation into osteocyte and adipose cells was investigated. Also, on the scaffolds, cytotoxicity and apoptosis were studied. To investigate the differentiation, treatment groups include: (1) BM-MSCs were plated in DMEM culture medium with high glucose containing 10% FBS and antibiotics (negative control); (2) BM-MSCs were plated in osteogenic differentiation medium (positive control); (3) positive control group + FeO-NPs, (4) positive control group + CuO-NPs; (5) BM-MSCs were plated in osteogenic differentiation medium on chitosan/alginate scaffold; (6) BM-MSCs were plated in osteogenic differentiation medium on chitosan/alginate/FeO-NPs scaffold; and (7) BM-MSCs were plated in osteogenic differentiation medium on chitosan/alginate/CuO-NPs scaffold. Alkaline phosphatase enzyme concentrations, mineralization rate using a calcium kit, and mineralization measurement by alizarin staining quantification were evaluated after 21 days of culture. In addition, qRT-PCR was used to assess the expression of the ALP, ColA, and Runx2 genes. When compared to other treatment groups, the addition of CuO-NPs in the chitosan/alginate hydrogel significantly increased the expression of the ColA and Runx2 genes (p < 0.05). However, there was no significant difference between the chitosan/alginate hydrogel groups containing FeO-NPs and CuO-NPs in the expression of the ALP gene. It appears that the addition of nanoparticles, in particular CuO-NPs, has made the chitosan/alginate scaffold more effective in supporting osteocyte differentiation.

Endoplasmic reticulum stress-dependent regulation of carboxypeptidase E expression in glioblastoma cells.

Objective. Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the CPE gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Methods. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the CPE gene was studied by quantitative RT-PCR and normalized to ACTB. Results. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of CPE gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the CPE gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of CPE gene in both types of used cells, but ERN1 inhibition enhanced this effect. Conclusion. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic CPE gene through protein kinase activity of ERN1 and that increased CPE gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.

Anti-inflammatory effects of pulsed magnetic field on the immune system

When an inflammatory response occurs in the body, primary immune cells, such as macrophages, phagocytize and present antigens, and secondary immune cells, such as T cells, produce antibodies against the presented antigens to prevent infection in the body. Abnormalities in the immune system releases pro-inflammatory cytokines such as TNF-α and IL-6, leading to rheumatoid arthritis and sepsis. This study aimed to confirm the change in inflammatory activity across innate immunity and acquired immunity while investigating the effects of pulsed magnetic field (PMF) on the immune system to regulate excessive inflammation. Raw 264.7 and EL4 cells were co-cultured in a DMEM medium using permeable cell culture inserts and inflammation was induced using LPS. Our PMF stimulator has the maximum intensity of 4700 G at a transition time of 136 μs with pulse intervals of 1 Hz. In order to confirm anti-inflammatory effects of PMF, the level of IL-6, pH and mitochondrial respiration rate (MRR) were measured using ELISA, pH meter and MTT assay, respectively. It was observed in vitro that inflammation-induced macrophages induce activation of T cells, as evidenced by the faster acidification in pH, increased IL-6 expression, and enhanced MRR, compared to T cells. But after PMF stimulation, pH levels improved, the concentration of IL-6 reduced, and MRR decreased in both macrophages and T-cells, which indicates inflammation relief. Our results show that macrophages, the primary immune cells, are first involved in the inflammatory response and recovery and influence T cells, the secondary immune cells. Therefore PMF stimulus is thought to affect the immune system of the human body by balancing the activation and suppression of immune cells and improve anti-inflammation. For clinical use, non-invasive PMF can be developed as a medical devices modulating immune system with optimizing the PMF conditions such as pulse shape, duration, or repetition rate.

The role of palbociclib on the alterations in CDKN2, CCNE1, E2F3, MDM2 expressions as target genes of miR-141.

According to WHO, Breast cancer is widely considered to be the first or second cause of cancer-related death almost universally. Cell cycle disruption, either in the form of uncontrolled expression of cyclins or because of the suspension in negative regulatory proteins (CDK inhibitors), was found to cause breast cancer. Palbociclib as specific CDK4/6 inhibitor is used for the treatment of ER+ metastatic cancers. In this study, we are looking to investigate the effect of palbociclib on breast cancer cells and evaluate the changes in the expression of some genes involved in the cell cycle as target genes of miR-141 after treatment with this drug. We used MCF7 as functional estrogen and non-invasive and MDA-MB-231 cell lines as triple-negative type of breast cancer and a model for more aggressive. MCF7 and MDA-MB-231 cell lines were cultured in DMEM medium. After counting cells and measuring viability, Palbociclib was administered at varying doses using the IC50 obtained from MTT, with the treatment given at two time points of 24 and 72 hours. RNA was extracted from untreated and treated cells and RNAs were converted to cDNA in the end. Gene expression changes were investigated by real-time PCR. Data management and analysis were conducted using GraphPad Prism 5.01 software. Among investigated genes, E2F3 gene was not significantly affected by Palbociclib in any of cell lines and time points. Besides, the expression of CCNE1 gene was significantly suppressed. It seems this drug was unable to reduce the expression of MDM2 gene significantly in triple negative (MDA-MB-231) cancer cells; however, a decrease was observed in luminal A (MCF-7) cells. CDKN2A and miR-141 genes expression increased significantly after treatment which can be aligned with palbociclib in proliferation inhibition.

Prostat Kanserinde Yağ Damlacık Oluşumu ile İlişkili PLA2G7, UCP2 ve NEDD4L Genlerinin Protein Ekspresyonlarının Araştırılması

Objective: Prostate cancer (PCa) is characterised by dysregulated lipid metabolism. PCa cells store lipids as lipid droplets and use them to facilitate proliferation and growth. In this study, we aim to investigate the expression levels of PLA2G7, UCP2 and NEDD4L proteins, which are involved in lipid droplet metabolism, in PC3 (advanced metastatic) and DU145 (intermediate metastatic) PCa cells. Materials and Methods: DU145 and PC3 cells were cultured in a high-glucose DMEM medium containing 10% FBS, 1% penicillin-streptomycin, and 1% non-essential amino acid. The expression levels of PLA2G7, UCP2, and NEDD4L protein were assessed with ELISA assay. Results: There was no significant difference in the protein level of PLA2G7 between the PC3 and DU145 cells (p&amp;gt;0.05), while the protein level of UCP2 increased in the PC3 cell line significantly (p&amp;lt;0.05). The protein level of NEDD4L decreased significantly in the DU145 cell line when compared to the PC3 cell line (p&amp;lt;0.05). Conclusions: As a result of this study, the UCP2 gene might play a role in the progression of prostate cancer, and there could be a relationship between NEDD4L and cell proliferation control.