Modified Eagle Research Articles

244 Fgf-21 Myokine Expression By Cultured Bovine Satellite Cells from 3- and 11-Month-Old Steers

Abstract Myokines are proteins produced by muscle cells that regulate muscle growth and metabolism. In livestock species, little is known about most myokines or their physiological impact. While FGF-21 has been examined in humans and model species for its roles in muscle development, regeneration, and metabolism, it is important to characterize its expression in livestock, such as beef cattle. This study assessed the quantity and temporal abundance of fibroblast growth factor 21 (FGF-21) expression, from cultured bovine satellite cells (BSC). BSC were isolated from the semimembranosus muscle of 3-month-old (n = 3) and 11-month-old (n = 3) commercially raised angus-based beef steers. BSC were cultured on Matrigel in Dulbecco’s Modified Eagle Medium with 10% Fetal Bovine Serum until 70% confluency. At this time, the media was replaced with fresh media representing time zero. Cells and media were collected at times 0-, 12-, 24-, 48-, 72-, and 96-hour with zero serving as the baseline. All experiments included three biological and three technical replicates. The content of myokines including FGF-21, were analyzed in the sample’s media using a commercial ELISA kit and cells were collected and stored for gene expression analysis. Statistical analysis was completed using the GLIMMIX procedure in SAS. A repeated measures analysis was used to evaluate the fixed effects of age, time, and age×time interaction. Contrasts were used to identify whether the baseline was statistically different from the other timepoints, and Tukey-Kramer adjustments were made. Significance was determined at P ≤ 0.05. There was an interaction effect between age and time (P < 0.0001). The media from the BSC of the 11-month-old steers had greater (P < 0.0001) FGF-21 concentrations than that of the 3-month-old steers at every time point. When evaluating each time in comparison with the baseline of each age independently, there were differences between 0 and 12 hour (P < 0.0001) and 0 and 24 hour (P = 0.002) for the 3-month-old steer BSC, as well as 0 and 12 hour (P = 0.0003) for 11-month-old steer BSC. This study confirms that the myokine FGF-21 is produced by cultured BSC, and in differing quantities depending on the age of the steer at harvest. The observed differences in FGF-21 production in cultured cells harvested from 3- to 11-month-old steers may in part be responsible for some of the differences in growth patterns in muscle cells at these ages. The authors postulate that these data represent the first report of FGF-21 expression in cultured bovine satellite cells.

Tuning the long-term corrosion behaviour of biodegradable WE43 magnesium alloy by PEO coating

Plasma electrolytic oxidation (PEO) coatings can control fast degradation of next-generation biodegradable magnesium alloys for biomedical applications. In this work, we studied the effect of a PEO coating on the long-term corrosion behaviour of medical-grade WE43 magnesium alloy. In vitro experiments were performed for periods of up to 4 weeks in an organic fluid Dulbecco's Modified Eagles' Medium (DMEM). The average corrosion rates were measured using the hydrogen evolution method, and the degradation of the mechanical integrity were quantified by tensile test. Susceptibility towards localized corrosion was evaluated by scanning electron microscopy (SEM) images of cross-sections. Moreover, energy-dispersive X-ray spectroscopy (EDS) mappings and X-ray diffraction (XRD) were utilised for analysing the corrosion layer. Experimental results reveal significant changes in the product layer formation due to the PEO coating in phosphate-based electrolyte. The porous morphology of the coating with an initial thickness of ∼20 ± 5 μm changed significantly during the corrosion process, and a growing Mg-O-rich layer of 32 μm after 28 days was detected at the substrate interface. In contrast to the non-coated control group, no carbon-enriched area was observed. These findings imply significant long-term corrosion protection of the PEO coating. The results provide valuable information for a better understanding of the in vitro degradation process in PEO-coated magnesium alloys.

Selective inhibition of indoleamine and tryptophan 2,3-dioxygenases: Comparative study on kynurenine pathway in cell lines via LC-MS/MS-based targeted metabolomics

In the last decade, the kynurenine pathway, which is the primary metabolic route for tryptophan (TRP) catabolism, has sparked great interest in the pharmaceutical sciences due to its role in immune regulation and cancer immunoediting. In this context, the development of cell-based assays might represent a tool to: i) characterize the cell secretome according to cell types; ii) gain more insight into the role of kynurenines in different disease scenarios; iii) screen hIDO1 (human indoleamine 2,3-dioxygenase) inhibitors and evaluate their effect on downstream TRP-catabolizing enzymes. This paper reports a validated Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify TRP, L-kynurenine (KYN), xanthurenic acid (XA), 3-hydroxykynurenine (3OHKYN), kynurenic acid (KA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid (AA), 5-hydroxytryptamine (serotonin, 5HT) and tryptamine (TRYP) in Dulbecco’s Modified Eagle and Eagle’s Minimum Essential Media (DMEM and EMEM, respectively). The quantitative method was validated according to FDA, ICH and EMA guidelines, later applied: i) to assess the impact of selective inhibition of hIDO1 or hTDO (human tryptophan 2,3-dioxygenase) on the kynurenine pathway in A375 (melanoma), MDA-MB-231 (breast cancer), and U87 (glioblastoma) cell lines using multivariate analysis (MVA); ii) to determine the IC50 values of both well-known (i.e., epacadostat, linrodostat) and the novel hIDO1 inhibitor (i.e., BL5) in the aforementioned cell lines. The proposed LC-MS/MS method is reliable and robust. Furthermore, it is highly versatile and suitable for applications in the preclinical drug research and in vitro assays.

Electrochemical, mechanical, and antibacterial properties of the AZ91 Mg alloy by hybrid and layered hydroxyapatite and tantalum oxide sol–gel coating

Abstract The corrosion and bacterial behavior of AZ91 magnesium alloy coated with sol–gel-deposited amorphous tantalum oxide and hydroxyapatite have been investigated. The objective was to assess the potential suitability of AZ91 for permanent prosthesis applications. The coatings were applied in layered and hybrid configurations and characterized using various techniques including X-ray diffractometry, Fourier transform infrared spectroscopy, scanning electron microscopy/energy dispersive spectrometry, and drop analyses. The antibacterial properties were evaluated through interactions with Staphylococcus aureus and Escherichia coli strains. Mechanical properties and adhesion were determined via linear scratch tests, and electrochemical corrosion tests were conducted in different media. The release of aluminum ions from the samples in Dulbecco’s Modified Eagle’s Medium was monitored over 28 days. The findings revealed that the amorphous tantalum oxide coating, particularly in combination with hydroxyapatite, improved antibacterial properties and positively influenced corrosion and scratch resistance. The layered and hybrid coatings demonstrated the highest corrosion resistance. The release of aluminum ions remained within acceptable levels in the tested medium. Overall, the study provides valuable insights into the potential of sol–gel coatings on AZ91 for prosthetic applications, considering antibacterial behavior, corrosion resistance, and aluminum release.

MDMB-FUBINACA Influences Brain Angiogenesis and the Expression of VEGF, ANG-1, and ANG-2.

This study aims to explore the impact of the synthetic cannabinoid methyl 2-(1-(4- fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (MDMB-FUBINACA) on the angiogenesis process in human brain microvascular endothelial cells. Synthetic cannabinoids (SCs) are substances that mimic the natural components found in the cannabis plant. SCs are considered prohibited substances that have a clear impact on the central nervous system (CNS). The purpose of this study is to explore how MDMB-FUBINACA influences angiogenesis in human brain microvascular endothelial cells and to clarify the pathways related to the cannabinoid receptors. Human brain microvascular endothelial cells (hBMECs) were grown in the medium containing Dulbecco Modified Eagle Medium (DMEM/F12) using an endothelial cell growth kit. Endothelial cell viability was evaluated using the MTT test. Migration ability was measured using the Wound healing test. The angiogenic capability was measured using a Tube Formation assay. Real-time polymerase chain reaction (RT-PCR) was utilized to explore the mRNA concentrations following MDMBFUBINACA treatment. ELISA and Western blotting were also employed to measure the protein levels. MDMB-FUBINACA greatly increases tube formation, endothelial cell proliferation, and migration. Pro-angiogenic factors such as angiopoietins 1 and 2 (ANG-1 and 2) and vascular endothelial growth factor (VEGF) were shown to be increased at both the RNA and protein levels. MDMB-FUBINACA induces the progression of the angiogenesis process by inducing the expression of pro-angiogenic factors. These findings aim toward developing novel treatments for angiogenesis- related disorders.

Procyanidin A1 improves sepsis-induced liver injury by inhibiting inflammation

Purpose: To determine the effect of procyanidin A1 (PCA1) on sepsis.Methods: Dulbecco’s Modified Eagle Medium (DMEM) was employed to incubate mouse hepatic cell line AML12. The AML12 cells treated with lipopolysaccharide (LPS, 50 μg/mL) was used to establish a sepsis cell model. Cell viability was evaluated using CCK-8 assay, while cell apoptosis was assessed by flow cytometry. Aspartate transaminase (AST), alanine aminotransferase (ALT), IL-6 and TNF-α levels were evaluated by enzyme linked immunosorbent assay (ELISA). Protein expressions were assessed using western blot assay.Results: The viability of AML12 cells decreased following treatment with IL-1β, but this change was offset by PCA1 treatment (40 or 80 μM). Similarly, cell apoptosis was enhanced after LPS treatment, but this change was attenuated by PCA1 treatment. The AST, ALT, IL-6 and TNF-α levels were all elevated after LPS treatment, but these changes were also reversed by PCA1 treatment, indicating that PCA1 suppressed LPS-induced liver injury and inflammation. Furthermore, the protein levels of p-p65/p65 and p-IκBα increased, and IκBα lowered following LPS treatment, but these effects were reversed by PCA1 treatment, indicating that PCA1 retarded NF-κB pathway.Conclusion: PCA1 alleviates sepsis-induced liver injury by inhibiting inflammation through NF-κB pathway. This suggestes that PCA1 may be an therapeutic agent for the treatment of sepsis.

Microstructure and Corrosion Behaviour of Mg-Ca and Mg-Zn-Ag Alloys for Biodegradable Hard Tissue Implants

Trauma orthopaedic surgery was the first domain to use degradable metallic implants made of magnesium alloys since the early 20th century. Unfortunately, the major limitation that consists of rapid degradation and subsequent implant failure, which occur in physiological environments with a pH between 7.4 and 7.6, prevents its widespread application. The biggest challenge in corrosion assay is the choice of the testing medium in order to reproduce more closely in vivo conditions. The current study was focused on two Mg-Zn-Ag alloys (Mg7Zn1Ag and Mg6Zn3Ag) and the Mg1Ca alloy. Dulbecco’s Modified Eagle Medium (DMEM) and Kokubo’s simulated body fluid solution (SBF) were selected as testing mediums and we follow the corrosion evaluation by the corrosion rate and mass loss. Also, the corrosion behaviour was interpreted in correlation with the microstructural features and alloying elements of the experimental magnesium-based alloys revealed by optical microscopy (OM), X-ray diffraction (XRD), and scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDX). The experimental results highlight the more corrosive nature of the SBF environment and that a higher percentage of silver (2.5 wt.%) exhibited a better corrosion resistance. We consider that the magnesium alloy Mg6Zn3Ag showed valuable biodegradation characteristics to be considered as raw materials for manufacturing small trauma implants.

γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus.

The association between diabetes mellitus (DM) and the increased risk and progression of cholangiocarcinoma (CCA) has been reported with unclear underlying mechanisms. Previous studies showed that γ-aminobutyric acid (GABA) B2 receptor (GABBR2) was upregulated in CCA cells cultured in high glucose (HG) conditions. Roles of GABA receptors in CCA progression have also been studied, but their association with DM and hyperglycemia in CCA remains unclarified. To investigate the effects of hyperglycemia on GABBR2 expression and the potential use of GABBR2 as a CCA therapeutic target. CCA cells, KKU-055 and KKU-213A, were cultured in Dulbecco Modified Eagle's Medium supplemented with 5.6 mmol/L (normal glucose, NG) or 25 mmol/L (HG) glucose and assigned as NG and HG cells, respectively. GABBR2 expression in NG and HG cells was investigated using real-time quantitative polymerase chain reaction and western blot. Expression and localization of GABBR2 in CCA cells were determined using immunocytofluorescence. GABBR2 expression in tumor tissues from CCA patients with and without DM was studied using immunohistochemistry, and the correlations of GABBR2 with the clinicopathological characteristics of patients were analyzed using univariate analysis. Effects of baclofen, a GABA-B receptor agonist, on CCA cell proliferation and clonogenicity were tested using the MTT and clonogenic assays. Phospho-kinases arrays were used to screen the affected signaling pathways after baclofen treatment, and the candidate signaling molecules were validated using the public transcriptomic data and western blot. GABBR2 expression in CCA cells was induced by HG in a dose- and time-dependent manner. CCA tissues from patients with DM and hyperglycemia also showed a significantly higher GABBR2 expression compared with tumor tissues from those with euglycemia (P < 0.01). High GABBR2 expression was significantly associated with a poorer non-papillary histological subtype but with smaller sizes of CCA tumors (P < 0.05). HG cells of both tested CCA cell lines were more sensitive to baclofen treatment. Baclofen significantly suppressed the proliferation and clonogenicity of CCA cells in both NG and HG conditions (P < 0.05). Phospho-kinase arrays suggested glycogen synthase kinase 3 (GSK3), β-catenin, and the signal transducer and activator of transcription 3 (STAT3) as candidate signaling molecules under the regulation of GABBR2, which were verified in NG and HG cells of the individual CCA cell lines. Cyclin D1 and c-Myc, the common downstream targets of GSK3/β-catenin and STAT3 involving cell proliferation, were accordingly downregulated after baclofen treatment. GABBR2 is upregulated by HG and holds a promising role as a therapeutic target for CCA regardless of the glucose condition.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Nitazoxanide is rapidly broken down to its active metabolite tizoxanide upon administration. Unlike many other candidates being investigated, tizoxanide plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbecco’s Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Herein a validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6 ng/mL to 1000 ng/mL. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

Comparison of the cytotoxicity of 3D-printed aligners using different post-curing procedures: an in vitro study

Abstract Objective Three-dimensional (3D) printing technology represents a novel method for manufacturing aligners. The aim of the present study was to assess the in-vitro cytotoxicity of 3D-printed aligners using different post-polymerisation conditions. Materials Aligners were printed using the same 3D-print resin (TC-85DAC, Graphy, Seoul, Korea) and printer (AccuFab-L4D, Shining 3D Tech. Co., Hangzhou, China), followed by different post-curing procedures. Six aligners were post-polymerised for 14 min using the Tera Harz Cure and a nitrogen generator curing machine (THC2, Graphy, Seoul, Korea) (P1). A further six aligners were post-cured for 30 min on each side using the Form Cure machine (FormLabs Inc, Somerville, USA) (P2). The aligners were cut into smaller specimens (2 mm×2 mm) and sterilised at 121°C. The specimens were placed in 96-well plates containing Dulbecco’s Modified Eagle’s Medium (DMEM) at 37° for 7 or 14 days. The viability of MC3T3E-1 pre-osteoblasts cultured with DMEM was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The optical density of each cell culture was measured to assess cell viability, following which the data were statistically analysed using two-way and one-way ANOVA (α = 0.05). Results The comparison of cytotoxicity revealed statistically significant differences between post-curing procedures and MTT timings (P &lt; 0.001). After 7 and 14 days, the cell viability of P2 was significantly reduced compared to P1 and the control groups (P &lt; 0.001), while P1 showed no significant differences compared to the controls. Overall, P2 post-curing exhibited moderate cytotoxicity, while P1 post-polymerisation was highly biocompatible. Conclusions Different post-curing procedures may affect the in-vitro cytotoxicity of 3D-printed aligners. Clinicians should adhere to the manufacturer’s recommendations when using 3D-print resin.

Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco’s Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by &gt;90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.

Comparison of Culture Media for In Vitro Expansion of Oral Epithelial Keratinocytes

Background: Expansion of OKF6/TERT-2 oral epithelial cells in vitro is important for studying the molecular biology of disease and pathology affecting the oral cavity. Keratinocyte serum-free medium (KSFM) is the medium of choice for this cell line. This study compares three media for OKF6/TERT-2 cultures: KSFM, Dulbecco’s Modified Eagle Medium/Nutrient Mixture of Hams F-12 (DMEM/F12), and a composite medium comprised of DMEM/F-12 and KSFM (1:1 v/v), referred to as DFK. The toxicological effects of electronic cigarette liquids (e-liquids) on OKF6/TERT-2 cells cultured in these media were also compared. Methods: Cells were cultured in KSFM, DMEM/F12, or DFK, and cellular morphology, growth, wound healing and the gene expression of mucins and tight junctions were evaluated. Additionally, cytotoxicity was determined after e-liquid exposures. Results: Switching from KSFM to DMEM/F12 or DFK 24 h post-seeding leads to typical cellular morphologies, and these cultures reach confluency faster than those in KSFM. Wound-healing recovery occurred fastest in DFK. Except for claudin-1, there is no difference in expression of the other genes tested. Additionally, e-liquid cytotoxicity appears to be amplified in DFK cultures. Conclusions: DMEM/F12 and DFK are alternative media for OKF6/TERT-2 cell culture to study the molecular biology of disease and pathology, provided cells are initially seeded in KSFM.

Bovine Rectoanal Junction In Vitro Organ Culture Model System to Study Shiga Toxin-Producing Escherichia coli Adherence.

Studies evaluating the interactions between Shiga toxin-producing Escherichia coli O157:H7 (O157) and the bovine recto-anal junction (RAJ) have been limited to either in vitro analyses of bacteria, cells, or nucleic acids at the RAJ, providing limited information. Alternatively, expensive in vivo studies in animals have been conducted. Therefore, our objective was to develop a comprehensive in vitro organ culture system of the RAJ (RAJ-IVOC) that accurately represents all cell types present in the RAJ. This system would enable studies that yield results similar to those observed in vivo. Pieces of RAJ tissue, obtained from unrelated cattle necropsies, were assembled and subjected to various tests in order to determine the optimal conditions for assaying bacterial adherence in a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Tissue integrity was assessed using cell viability, structural cell markers, and histopathology, while the adherence of bacteria was evaluated via microscopy and culture methods. DNA fingerprinting verified the recovered bacteria against the inoculum. When the RAJ-IVOC was assembled in Dulbecco's Modified Eagle Medium, maintained at a temperature of 39 °C with 5% CO2 and gentle shaking for a duration of 3-4 h, it successfully preserved tissue integrity and reproduced the expected adherence phenotype of the bacteria being tested. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.

Impact of Canine Amniotic Mesenchymal Stem Cell Conditioned Media on the Wound Healing Process: In Vitro and In Vivo Study.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco's Modified Eagle's Medium-32.55%) and the positive control (Dulbecco's Modified Eagle's Medium supplemented with FGF2, N2, B27, and EGF-82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

Studies of New Layer Formation on the Surface of Zinc Doped Hydroxyapatite/Chitosan Composite Coatings in Biological Medium

Usually, before being used in biomedical applications, a biomaterials’ bioactivity is tested by in vitro methods that simulate similar conditions to those found in the human body. In this work, we report on the synthesis of zinc-doped hydroxyapatite–chitosan (ZnHApC) composite coatings by the vacuum deposition method. The surface microstructure and the chemical and molecular modification of the coatings before and after soaking in DMEM (Dulbecco’s Modified Eagle’s Medium) were studied. For this objective, techniques such as attenuated total reflection (ATR), Fourier transform infrared (FTIR) spectroscopy, metallographic microscopy (MM), and scanning electron microscopy (SEM) were applied used. Also, water contact angle measurements and swelling studies were made on ZnHApC composite coatings before and after soaking in a biological medium. The coatings’ adherence to the substrate was also studied. The results of antifungal studies on ZnHApC composite coatings against the Candida albicans microbial strain reveal their good antifungal activity. The biocompatibility of the composite coatings was tested using a primary osteoblast cell line. Our results suggest that zinc-doped hydroxyapatite–chitosan samples could be used as a bioimplant material due to their enhanced bioactivity and biocompatibility.

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer.

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco's Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38-24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer's disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines.

Propofol-Induced miR-493-3p Inhibits Growth and Invasion of Gastric Cancer through Suppression of DKK1-Mediated Wnt/β-Catenin Signaling Activation.

Gastric cancer (GC) is one of the most common malignant tumors and also one of the most deadly tumors. In recent years, studies have shown that propofol can inhibit the proliferation and metastasis of many tumor cells. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of GC cells. Human gastric cancer cell line SGC-7901 and human normal gastric epithelial cell GES-1 were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Propofol of different concentrations (0, 2, 5, and 10 μg/mL) was used to treat SGC-7901, and miR-493-3p inhibitor was transfected into SGC-7901. The cell proliferation of SGC-7901 was analyzed by MTT as well as colony formation assay. The qRT-PCR was used to assess the expression of mRNA for key genes. We examined the protein expression of DKK1 and relative markers with western blot. Putative binding places of miR-493-3p on the 3'-untranslated area of DKK1 were predicted using bioinformatics and dual-luciferase method. Propofol prohibited phenotypic features of GC, according to our findings. Furthermore, research into the underlying mechanisms of propofol's suppressive effects in GC cell proved that propofol therapy improved the degrees of expression of the potential tumor suppressor miR-493-3p. The inhibiting properties of propofol on GC cell development, migration, and invasion were abolished when propofol-induced miR493-3p was silenced with anti-miR-493-3p. We also found that this drug reversed epithelial-mesenchymal transformation in SGC-7901 cells via inducing miR-493-3p. Propofol-induced miR-493-3p decreases GC cell development via targeting DKK1 and hence inhibits Wnt/β-catenin signaling, according to these findings. Propofol-induced miR-493-3p decreased GC cell development via targeting DKK1 and hence inhibited Wnt/β-catenin signaling, according to these findings.

Using macrophage cell culture to study the processes that result in removal of blood from bruises

Macrophages are responsible for removing the extravasated blood that forms a bruise. Aspects of this process, such as how long it takes for erythrocytes to be phagocytosed and for iron to be demonstrated using Perls stain, are not established. This work sought to assess the possibility of using macrophages in cell culture to address some of the unknowns in the processes of resolution of a bruise. Human peripheral blood monocytes were obtained from buffy coats provided by the Australian Red Cross Blood Service. Monocytes were isolated by adherence to tissue culture plastic and differentiated into macrophages. Human blood clot was added with Dulbecco’s Modified Eagle Medium (DMEM) in combination with Fetal Calf Serum (FCS) and/or Heat inactivated human serum (HI HUS). After 24 hours the blood in the supernatant was washed off and replaced with fresh medium. It was noted red blood cells could be seen in macrophages. After 4 days, the macrophages showed prominent blue colour with Perls stain indicating the presence of hemosiderin. This pilot study has shown that in a cell culture system, macrophages can breakdown erythrocytes to produce hemosiderin. This model system may be used to investigate the processes behind resolution of bruises.

Analysis of Pulp Tissue Viability and Cytotoxicity of Pulp Capping Agents.

The present research study assessed the cell viability and cytotoxic effect of mineral tri-oxide aggregate (MTA), Tetric N-Bond Universal bonding agent, Theracal PT (pulpotomy treatment), and platelet-rich fibrin (PRF) as pulp capping agents on human dental pulp stem cells (hDPSCs). The cells were isolated from the pulp tissue of an extracted healthy permanent third molar. After four passages in Dulbecco’s Modified Eagle’s Medium, the primary cells were employed for the investigation. The test materials and untreated cells (negative control) were subjected to an Methylthiazol-diphenyl-tetrazolium (MTT) cytotoxicity assay and assessed at 24-, 48-, and 72-h intervals. The Wilcoxon matched-paired t-test and Kruskal−Wallis analysis of variance (ANOVA) test were applied (p < 0.05). PRF imparted the highest cell viability at 48 h (p < 0.001), followed by MTA, Theracal PT, and Tetric N-Bond. Similarly, PRF had the highest potential to enhance cell proliferation and differentiation (p < 0.001), followed by Theracal PT, MTA, and the bonding agent at the end of 24 h and 72 h, respectively. Finally, PRF sustained the viability of human primary dental pulp stem cells more effectively than Theracal PT and MTA; however, the application of a Tetric N-Bond as a pulp capping agent was ineffective.

Spent culture supernatant of Streptococcus gordonii mitigates inflammation of human periodontal cells and inhibits proliferation of pathogenic oral microbes.

The oral commensal bacterial species Streptococcus gordonii has been reported to regulate the inflammation of oral epithelial cells stimulated by the periodontal pathogen Porphyromonas gingivalis. This study investigated the activities of S. gordonii metabolites in S. gordonii spent culture supernatants (Sg-SCS) on periodontal-related bacterial growth and periodontitis-associated inflammatory cytokines. Sg-SCS was collected from S. gordonii cultures grown in Dulbecco Modified Eagle Medium and added to the growth media of representative health- and disease-related oral species: S. gordonii, Streptococcus sanguinis, Streptococcus mitis, Streptococcus oralis, P. gingivalis, Tannerella forsythia, and Treponema denticola. The Sg-SCS was also tested for its ability to regulate the expression of proinflammatory cytokines by human macrophages, epithelial cells, and gingival fibroblasts upon stimulation with P. gingivalis-derived lipopolysaccharide (Pg-LPS). Sg-SCS significantly reduced transcript and protein levels of interleukin (IL)-1β, 6, and 8 induced by Pg-LPS stimulation in multiple types of periodontal cells. mRNA sequencing and bioinformatics analyses indicated that Sg-SCS significantly affects 10 inflammatory pathways. Additionally, Sg-SCS exhibited suppression of the growth of periodontal disease-related bacteria, including T. denticola and P. gingivalis, along with the primary plaque-colonizing species S. oralis. At a low concentration, Sg-SCS also inhibits P. gingivalis adhesion. These results strongly suggest that S. gordonii-derived SCS contains metabolites that have anti-inflammatory properties and an ability to inhibit periodontitis-associated pathogenic bacteria. Further investigation will be needed to identify the individual metabolites within the Sg-SCS to develop a novel metabolite-based approach to treating and preventing periodontitis.