Modified Eagle Research Articles

The Thermal Stability of Influenza Viruses in Milk

Highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype (clade 2.3.4.4b) have been detected in raw milk from infected cows. Several studies have examined the time and temperature parameters to ascertain whether influenza viruses in milk can be inactivated completely under commercial pasteurization conditions, yielding conflicting results. This study aimed to investigate whether milk could help protect influenza viruses from heat treatment. After heat treatment at 49 °C for one hour, the titer reduction of the influenza A/WSN/1933 (A/H1) virus in milk was approximately 1.6 log10TCID50/mL, which was significantly lower than that (3 log10TCID50/mL) observed in the Dulbecco’s Modified Eagle Medium (DMEM) control media. The influenza D/bovine/CHN/JY3002/2022 (D/Yama2019) virus in milk retained a high residual infectivity (4.68 × 103 log10TCID50/mL) after treatment at 53 °C; however, the virus in DMEM completely lost its infectivity under the same conditions. Moreover, the influenza A/chicken/CHN/Cangzhou03/2023 (A/H5) virus in DMEM could be inactivated completely using any of the three heat treatment methods: 63 °C for 30 min, 72 °C for 15 s, or 80 °C for 15 s. For the virus present in milk, only heat treatment at 80 °C for 15 s completely inactivated it. These results suggest that milk prevents influenza viruses from pasteurization inactivation.

Mechanical Properties of 3D Printed PLA Scaffolds for Bone Regeneration

Abstract The growing interest in biodegradable scaffolds for bone regeneration created a need to investigate new materials suitable for scaffold formation. Poly(lactic acid) (PLA) is a polymer commonly used in biomedical engineering, e.g. in tissue engineering as a biodegradable material. However, the mechanical behavior of PLA along its degradation time is still not explored well. For this reason, the mechanical properties of PLA scaffolds affected by incubation in physiological medium needs to be investigated to show the potential of PLA to be used as a material for biodegradable scaffold formation. The purpose of this research is to determine the mechanical properties of PLA scaffolds before and after incubation, and to apply constitutive material models for further behavior prediction. Two sets of PLA scaffolds were printed by the 3D printer “Prusa i3 MK3S” and sterilized by ultraviolet light and ethanol solution. The first set of specimens was incubated in DMEM (Dulbecco’s Modified Eagle Medium) for 60, 120, and 180 days maintaining 36.5 °C temperature. The mechanical properties of the scaffolds were determined after performing the compression test in the “Mecmesin MultiTest 2.5-i” testing stand with a force applied at two different speed modes. The obtained data was curve fitted with the hyperelastic material models for a model suitability study. The second set of specimens was incubated in PBS (Phosphate Buffered Saline) for 20 weeks and used in a polymer degradation study. The obtained results show that the mechanical properties of PLA scaffolds do not decrease during incubation in physiological medium for a predicted new bone tissue formation period, though hydrolysis starts at the very beginning and increases with time. PLA as a material seems to be suitable for the use in bone tissue engineering as it allows to form biocompatible and biodegradable scaffolds with high mechanical strength, required for effective tissue formation.

The Utilization of Central Composite Design for the Production of Hydrogel Blends for 3D Printing

Central composite design (CCD) is a statistical experimental design technique that utilizes a combination of factorial and axial points to study the effects of multiple variables on a response. This study focused on optimizing hydrogel formulations for 3D printing using CCD. Three biopolymers were selected: sodium alginate (SA), gelatin (GEL), and carboxymethyl cellulose (CMC). The maximum and minimum concentrations of each polymer were established using a Google Scholar search, and CCD was employed to generate various combinations for hydrogel preparation. The hydrogels were characterized in accordance with their swelling degree (SD) in phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagle Medium (DMEM), as well as their printability in 2D and 3D assays. The formulation consisting of 7.5% SA, 7.5% GEL, and 2.5% CMC exhibited the best swelling properties and exceptional printability, surpassing all other tested formulations. This study highlights the effectiveness of design of experiment methodologies in accelerating the development of optimized hydrogel formulations for various applications in 3D printing and suggests avenues for future research to explore their performance in specific biological contexts.

Phenotypic characteristics of adipocyte-like cells generated from C2C12 myoblasts cultured with chicken serum

The aim of this study was to clarify the transcriptional and metabolic characteristics of C2C12 myoblasts cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% chicken serum (CHS) (C2C12-CHS cells) compared with C2C12 myoblasts cultured in DMEM containing 20% fetal bovine serum (FBS) (C2C12-FBS cells). After 3 days of culture, C2C12-CHS cells showed a marked accumulation of lipid droplets, accompanied by increased expression levels of brown adipocyte-related genes (i.e., Bmp7, Prdm16, Ucp1, Cidea, Pgc1α, Cox7a1, Cox8, and β3-adorenoceptor). Furthermore, stimulation of β3-adorenoceptor by its selective agonist, mirabegron, increased the mRNA expression of Ucp1 and Pgc1α in C2C12-CHS cells. Wide-targeted metabolomic analysis performed by gas chromatography-tandem mass spectrometry revealed that the metabolic profile of C2C12-CHS cells was obviously different to that of C2C12-FBS cells. Additionally, the metabolomic analysis indicated that β3-adrenoceptor stimulation by mirabegron upregulated energy metabolism in C2C12-CHS cells as seen in brown adipocytes. These results suggest that C2C12-CHS cells may differentiate into brown adipocyte-like cells, accompanied by increased functional β3-adrenoceptor.

Effects of Pressure, Hypoxia, and Hyperoxia on Neutrophil Granulocytes

Background: The application of normo- and hyperbaric O2 is a common therapy option in various disease patterns. Thereby, the applied O2 affects the whole body, including the blood and its components. This study investigates influences of pressure and oxygen fraction on human blood plasma, nutrient media, and the functions of neutrophil granulocytes (PMNs). Methods: Neutrophil migration, reactive oxygen species (ROS) production, and NETosis were examined by live cell imaging. The treatment of various matrices (Roswell Park Memorial Institute 1640 medium, Dulbecco’s Modified Eagle’s Medium, H2O, human plasma, and isolated PMNs) with hyperbaric oxygen (HBO) was performed. In addition, the expression of different neutrophil surface epitopes (CD11b, CD62L, CD66b) and the oxidative burst were investigated by flow cytometry (FACS). The application of cold atmospheric plasma (CAP) to normoxic and normobaric culture media served as a positive control. Soluble reaction products such as H2O2, reactive nitrogen species (RNS: NO2− and NO3−), and ROS-dependent dihydrorhodamine oxidation were quantified by fluoro- and colorimetric assay kits. Results: Under normobaric normoxia, PMNs migrate slower and shorter in comparison with normobaric hyper- or hypoxic conditions and hyperbaric hyperoxia. The pressure component has less effect on the migration behavior of PMNs than the O2 concentration. Individual PMN cells produce prolonged ROS at normoxic conditions. PMNs showed increased expression of CD11b in normobaric normoxia, lower expression of CD62L in normobaric normoxia, and lower expression of CD66b after HBO and CAP treatment. Treatment with CAP increased the amount of ROS and RNS in common culture media. Conclusions: Hyperbaric and normobaric O2 influences neutrophil functionality and surface epitopes in a measurable way, which may have an impact on disorders with neutrophil involvement. In the context of hyperbaric experiments, especially high amounts of H2O2 in RPMI after hyperbaric oxygen should be taken into account. Therefore, our data support a critical indication for the use of normobaric and hyperbaric oxygen and conscientious and careful handling of oxygen in everyday clinical practice.

Roles of Prostaglandins and Hydrogen Sulfide in an Outflow Model of the Porcine Ocular Anterior Segment Ex Vivo

Background: Hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive rabbits by increasing aqueous humor (AH) outflow through the trabecular meshwork. In the present study, we investigated the contribution of endogenous H2S and the role of intramurally generated prostaglandins in the observed increase in AH outflow facility in an ex vivo porcine ocular anterior segment model. Material and Methods: Porcine ocular anterior segment explants were perfused with Dulbecco’s Modified Eagle’s Medium maintained at 37 °C and gassed with 5% CO2 and 95% air under an elevated pressure of 15 mmHg for four hours. Perfusates from the anterior segment explants were collected and immediately assayed for their H2S and prostaglandin E2 content. Results: Elevating perfusion pressure from 7.35 to 15 mm Hg significantly (p < 0.001) increased H2S concentration in the perfusate from 0.4 ± 0.1 to 67.6 ± 3.6 nM/µg protein. In the presence of an inhibitor of cystathionine ß-synthase/cystathionine γ-lyase, aminooxyacetic acid (AOAA, 30 µM), or an inhibitor of 3-mercaptopyruvate sulfurtransferase, α-ketobutyric acid (KBA, 1 mM), the effects of elevated pressure on H2S levels in the perfusate was significant (p < 0.001). Furthermore, flurbiprofen (30 µM) and indomethacin (10 µM) attenuated the elevated pressure-induced increase in H2S levels in the perfusate. Interestingly, elevating perfusion pressure had no significant effect on PGE2 concentrations in the perfusate. While the inhibition of H2S biosynthesis by AOAA or KBA did not affect PGE2 levels in perfusate, flurbiprofen (30 µM) caused a significant (p < 0.05) decrease in the concentration of PGE2 under conditions of elevated perfusion pressure. Conclusions: We conclude that the elevated perfusion pressure-induced increase in H2S concentrations depends upon the endogenous biosynthesis of H2S and intramurally produced prostaglandins in the porcine anterior segment explants. While the concentration of PGE2 in the perfusate under elevated perfusion pressure was unaffected by pretreatment with inhibitors of H2S biosynthesis, it was reduced in the presence of an inhibitor of cyclooxygenase.

PSIV-6 Blood biomarkers of oxidative stress in offspring from dams infected with bovine viral diarrhea virus during late-term gestation

Abstract The objective of this experiment was to examine the impact of dams infected with bovine viral diarrhea virus (BVDV) during late gestation on inflammatory blood biomarkers in their offspring. Fetal infection with BVDV before d 125 to 150 of gestation results in the birth of immunotolerant, persistently infected (PI) calves. Infection of BVDV during late gestation results in transient fetal infections (TI). It was hypothesized that offspring of dams transiently infected with BVDV on d 175 of gestation would have greater inflammatory blood biomarkers than control offspring. Unvaccinated, yearling Hereford heifers (n = 24), seronegative for antibodies to BVDV1 and BVDV2 were bred by artificial insemination with X chromosome-bearing sperm from an Angus sire. On d 175 of pregnancy, heifers were intranasally inoculated with either Dulbecco’s Modified Eagle Medium + 2% horse serum (sham control) or 4.0 log TCID50 noncytopathic type 2 BVDV to generate control and transient infected calves, respectively. All BVDV-inoculated dams seroconverted by d 14 post-inoculation and all sham-inoculated control dams remained seronegative. Twelve, seronegative control heifer calves were born to the control dams, and 11 seropositive (to type 2 BVDV) TI heifer calves were born to BVDV-inoculated dams. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our feedlot research facility, housed in one group feedlot pen, and transitioned to a high-energy concentrate-based diet until reaching an approximate body weight (BW) of 600 kg. Upon arrival, all animals received a standard heifer growth implant containing 200 mg of testosterone propionate and 20 mg of estradiol benzoate, a modified live viral vaccine containing IBR-BRSV-PI3, and were dewormed. Jugular blood samples were obtained every 28 d until harvest (280 d on feed) and analyzed for biomarkers of inflammation. Data were analyzed as a randomized block design with the MIXED procedures of SAS. There was a treatment x time interaction (P < 0.05) for plasma ceruloplasmin and the oxidized form of glutathione. On d 0 and 28 of the feeding period, all biomarkers were similar across treatments. By d 56 of the feeding period, ceruloplasmin (P < 0.03) and the oxidized form of glutathione (P < 0.01), indicators of chronic inflammation, were increased in plasma samples from TI heifers compared with controls. These parameters remained greater in TI heifers compared with controls throughout the feeding period. There were no treatment or treatment x time interactions for plasma interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and haptoglobin. These data suggest that TI heifers may be experiencing a greater level of oxidative stress later in the finishing period. This research was supported by USDA-NIFA Grant #2019-67015-29866.

Three-dimensional Culture of Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

Background: The mammary gland is a crucial organ in dairy animals, responsible for milk production, regulated by intricate hormonal and cellular interactions. Traditional two-dimensional (2D) cell culture systems used in mammary gland research fail to replicate the complex in vivo environment, limiting the understanding of mammary gland biology. The study explores the advantages of three-dimensional (3D) culture systems for Buffalo Mammary Epithelial Cells (BuMECs), aiming to enhance morphological and functional differentiation compared to 2D cultures. Methods: BuMECs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 10% FBS and specific growth factors. For 2D culture, BuMECs were grown on a plastic substratum. For 3D culture, cells were cultured on top of and embedded in growth factor-reduced (GFR) Matrigel. Morphological differentiation was observed under phase-contrast microscopy. Total RNA was extracted and cDNA was synthesized to measure β-casein gene expression using quantitative real-time PCR (qPCR). Protein expression was analyzed by Western blotting, targeting total casein. Result: BuMECs grown in 3D cultures exhibited enhanced morphological differentiation, forming alveolar and ductal structures, unlike the monolayer formation observed in 2D cultures. Rapid formation of duct-like structures was noted within 24 hours in 3D cultures. Functional differentiation was significantly improved, evidenced by a five-fold increase in β-casein mRNA expression and higher protein levels of α, β and κ-casein in 3D cultured cells compared to 2D cultures. Western blot analysis confirmed the elevated casein protein expression in 3D cultures.

In Vitro Evaluation of Cytotoxicity of Moringa oleifera Hydroalcoholic Leaf Extract on Human Gingival Fibroblasts

Background. The market for wound healing biomaterials is a rapidly growing industry globally. Moringa oleifera Lam. (MO) is a plant that has gained attention for its benefit in buccal mouthwashes and antiseptic products. This study aimed to assess the cytotoxic potential of MO hydroalcoholic extract on human primary gingival fibroblasts. Methods. The gingival tissue was collected from a healthy adult male at Umm Al-Qura University Dental Teaching Hospital. Gingival fibroblasts were isolated and cultured in Dulbecco’s Modified Eagle Medium (DMEM). MO leaves were dried, powdered, and extracted with 80% ethanol. Various concentrations of MO alcohol extract were used to assess the cell viability of gingival fibroblasts using the MTT assay. Results. The gingival fibroblasts treated with higher concentrations of MO hydroalcoholic extract (10, 5, and 2.5 mg/ml) showed significant morphological changes, including cytoplasm swelling, ruptured cells, nuclear changes, and apoptotic bodies. At these concentrations, cell viability decreased sharply. However, at a lower concentration of 2.5 mg/ml, the cells showed better viability. MO alcohol extract at concentrations of 1.25 mg/ml or lower did not have cytotoxic effects on fibroblasts, with cell viability comparable to that of the control. Conclusions. MO alcohol extract at concentrations of 1.25 mg/ml or lower is not cytotoxic and does not induce dystrophic changes in morphology. Further research is needed to understand MO’s impact on oral health and to assess its potential use in oral care products in vivo.

Survival and growth of M. perstans larvae in a human colon carcinoma cell line-based in vitro culture

Mansonella perstans infections are widespread in Sub-Saharan Africa and Central and South America and thus can be considered as the most prevalent parasite of man in tropical Africa. In contrast to the high prevalence, knowledge about the biology of this filarial nematode is restricted and no effective treatment regimens of this ivermectin-resistant parasite is lacking. An obstacle for the research is that M. perstans resides in body cavities and thus have been only rarely recovered during surgery or autopsy. Therefore, alternative methods like in vitro culture systems need to be implemented to decipher the nature of mansonellosis and effective drugs. Previously, we have established a monkey kidney epithelial cell-based in vitro culture for the maintenance of M. perstans infective larvae (L3) up to 77 days. However, no alternative for this culture system have been postulated to allow longer survival rates and development of adult worms in vitro. Thus, we aim to establish an alternative in vitro culture system for M. perstans L3. M. perstans L3 were isolated from engorged and laboratory reared Culicoides midges. The larvae were then cultured in Dulbecco's Modified Eagle Medium supplemented with either 10% foetal bovine serum (FBS), 10% newborn calf serum (NCS) or 1% bovine serum albumin (BSA) together with human colon carcinoma cells (HCT-8) as feeder cells. Survival and growth were recorded. We obtained that the 10% NCS culture condition was superior allowing long-term maintenance of M. perstans L3 for up to 100 days and boosted growth of the parasites for up to 5-folds compared to the initial size at culture inception. Although no moulting of the L3 into L4 or adult worms could be overserved, the human colon carcinoma cell-based in vitro culture provides an alternative platform to analyse M. perstans biology and screen for novel drugs against M. perstans.

Effect of Experimental Parameters on the Electrochemical Current during Scanning Electrochemical Microscopy of Living Cells

Scanning Electrochemical Microscopy (SECM) is gaining popularity for its application to live cell imaging to monitor electrochemical processes occurring at the interface of living cells and electrodes. [1] Although many studies have been reported varying the instrumental parameters and using various probe designs, [2] the influence of experimental parameters on the electrochemical current must not be neglected, in order to generate reliable results. Our work explores parameters, such as temperature, light exposure and cell media during the SECM analysis of Adenocarcinoma Cervical cancer (HeLa) cells. The experimental and numerical studies with temperature variation revealed that temperature changes of 2ᵒC influence the reaction kinetics of single living cells. [3] Further, it was observed during light exposure that the electrochemical current becomes stabilized after 20 minutes of constant light environment compared to dark, where the standard deviation of the electrochemical current showed an increase in trend overtime. Furthermore, electrochemical current responses of HeLa cells in Phosphate Buffered Solution (PBS), Dubelcco’s Modified Eagle Medium (DMEM) and DMEM with 10% Fetal Bovine Serum (FBS) is being explored. These results will help to understand the origin of potential electrochemical current changes and the ideal experimental conditions to perform SECM measurements with living cells.[1] I. Beaulieu, S. Kuss, J. Mauzeroll, M. Geissler, Biological scanning electrochemical microscopy and its application to live cell studies, Anal. Chem. 83 (2011) 1485–1492. https://doi.org/10.1021/ac101906a.[2] D. Polcari, P. Dauphin-Ducharme, J. Mauzeroll, Scanning Electrochemical Microscopy: A Comprehensive Review of Experimental Parameters from 1989 to 2015, Chem. Rev. 116 (2016) 13234–13278. https://doi.org/10.1021/acs.chemrev.6b00067.[3] N. Thomas, D. Lima, D. Trinh, S. Kuss, Temperature Effect on the Electrochemical Current Response during Scanning Electrochemical Microscopy of Living Cells, Anal Chem. 95 (2023) 17962-17967. https://doi.org/10.1021/acs.analchem.3c03716

Bioactive coatings on biopolymer materials: evaluation of mechanical, physical, thermal, and in vitro properties

The aim of this study was to develop and characterize coatings of bioglass nanoparticles (BGNs) on biopolymer (poly [glycolide-co-L-lactide], (PGLA)) surgical sutures, and to investigate the effects of these coatings on the performance of the sutures as they slid through a skin substitute. Melt-derived BGNs were used to coat resorbable PGLA biopolymers, providing them with bioactivity, biocompatibility, and improved physical and mechanical properties. The structural, thermal, and physical properties of the coated and uncoated biopolymers were analyzed using Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FE-SEM) and Energy-Dispersive X-ray Spectroscopy (EDXS). The dissolution profiles and bioactivity of the BGNs-coated PGLA biopolymers were assessed through Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). Tensile strength tests were conducted on the biopolymers before and after immersion in simulated body fluid to evaluate the impact of the BGNs coating on the degradation of PGLA biopolymers. Incorporating BGNs into PGLA resulted in improved tensile strength properties. The study also found that increasing the BGNs ions content facilitated the formation of a hydroxycarbonate apatite (HCA) layer in Dulbecco’s Modified Eagle Medium and medium with L-glutamine and sodium bicarbonate solutions. In vitro bioactivity tests demonstrated that the coated suture biopolymers exhibited enhanced attachment, migration, and proliferation of fibroblasts, indicating favorable biocompatibility of the biomaterial for clinical applications.

Cyclic strain promotes MCP-1 expression in Dahl Salt Sensitive (SS) rat vascular smooth muscle cells (VSMCs) through the TGF-β pathway

Prior studies have suggested a role for TGF-β in the generation of hypertension in SS rats. We have shown that production of MCP-1 (CCL-2) and TGF-β1 increase in the vasculature of hypertensive SS rats. This study aims to explore novel mechanisms of hypertension development. We hypothesize that TGF-β1 regulated the production of MCP-1 by VSMCs in the setting of cyclic strain. Primary cultures of SS aortic VSMCs in early (<6) passages were grown in standard fashion in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. Cultured cells were identified as smooth muscle cells by their characteristic morphology and positive immunostaining for α-smooth muscle actin. In the first study, VSMCs were treated with recombinant active TGF-β1 (1 ng/mL and 10 ng/mL) for 6 h, then harvested for MCP-1 mRNA quantification by SYBR green qPCR. Medium was also collected to determine MCP-1 levels using ELISA. TGF-β1 increased MCP-1 mRNA expression by 2.0- and 3.1-fold in VSMC incubated in medium containing 1 ng/mL and 10 ng/mL of TGF-β1, respectively. In a second series of studies, VSMCs were seeded onto 6-well BioFlex plates. Some cells were pre-treated with 10 μmol/mL of an ALK4/5 inhibitor. Cyclic strain, an in vitro model of hypertension, was performed using the FlexCell 6000 and a standardized regimen (8 h, 15% elongation @ 1 Hz). Cells and medium were then harvested for mRNA analyses and determination of MCP-1 levels. When compared with expression by VSMCs maintained under static condition, cyclic strain increased (p<0.05) MCP-1 mRNA expression 4.2-fold and TGF-β1 mRNA 1.93-fold. Cyclic strain also increased (p<0.01) MCP-1 protein in the medium (static condition vs cyclic strain, 112.6 ± 7.1 pg/mL vs 222.5 ± 6.5 pg/mL, n=6). Pre-treatment of VSMC with the ALK4/5 inhibitor reduced mRNA expression of MCP-1 mediated by cyclic strain (Cyclic strain vs Cyclic strain + AKL4/5 inhibitor, 4.5 ± 1.06 vs 2.88 ± 0.47, n=6, P < 0.01). Thus, cyclic strain promotes MCP-1 production by VSMCs through the TGF-β pathway. The data support the need for further study of this interaction as well as a potential role in arterial hypertension in this rodent model. Department of Veterans Affairs Basic Sciences R&D (BSRD) Service (1 I01 BX005640) National Institutes of Health (P30 DK079337). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3048: The Role Of 17:0(2r-oh) Ceramide In Lps Activated Macrophages

Macrophages play a crucial role in the initiation and progression of atherosclerosis, contributing to the buildup of fatty deposits which results in the plaque formation in the walls of arteries. Macrophages modulate the mediators of inflammation and immune response in atherosclerosis. Bacterial lipopolysaccharide (LPS) exerts significant effects on these macrophages including secretion of pro-inflammatory cytokines. The current study is designed to examine the role of sphingosine intermediate N-(2'-(R)-hydroxyheptadecanoyl)-D-erythro-sphingosine (17:0(2R-OH) in the macrophage’s inflammation potential. Couple of studies have indicated a protective role for sphingosine however 17:0(2R-OH) didn’t receive adequate attention. Study design: Mouse J774 macrophages were cultured in Dulbecco’s Modified Eagle’s medium with 4.5g/L glucose and sodium pyruvate without L-glutamine containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C. The cells were treated in 6-well plates at a density of 5.5х10 6 cells/well. Sphingosine was dissolved in DMSO prior to the treatment. The cells were treated with the 17:0(2R-OH) with 3 different concentrations (5, 25, 50 μg) in duplicates with or without LPS except for the controls. The cells and media were collected at 3 time points (12,24,36) hours. Cells were harvested in Trizol reagent, RNA was then extracted from cells and genes expression were determined in RTPCR using SYBR Green. Results and Conclusion: Our initial microscopic observations suggest that 17:0(2R-OH) potentially modulates inflammation in macrophages, this appears to be dose-dependent. 17:0(2R-OH) has been identified in marine and freshwater fish, marine oils, seaweed, sea urchins, sea cucumbers and may have dietary benefits.

Effect of Different Processing Temperature on Motility and Viability in Canary Sperm

Background: Canaries have attracted people’s attention with their colors, shapes and impressive songs. The fact that canaries are domesticated and easy to obtain has made these birds attractive for songbird studies. In our study, canary semen was examined at different temperature values. This study aims to determine the most efficient spermatozoa examination temperature in canaries. Methods: In this study, five different temperature values (36, 38, 40, 42 and 44°C) were determined. The study used 6 active male Gloster canaries in each group. After each semen collection application, a 3-day break was given for the birds to rest. Dulbecco’s Modified Eagle Medium (DMEM) expander, which is preferred by songbirds, was used in the research. The semen was diluted 1:2 in a water bath at specified temperatures (36, 38, 40, 42, 44°C) and immediately examined for motility and viability. Result: When the study results were evaluated, it was seen that the viability rate at 38°C was higher than the other groups and 40°C gave the highest results in terms of motility. In addition to the viability rate at 38°C, the swimming speed of spermatozoa was also found to be more stable over a wider area than other groups. As a result, based on the data obtained from the study, it can be said that the most appropriate temperature for motility and viability in canary spermatozoa is 38°C.

A186 INFLAMMATION MODIFIES DOSE-DEPENDENT RESPONSES OF INTESTINAL ANTI-TUMOUR MICRORNAS TO CRANBERRY PROANTHOCYANIDIN AND ITS MICROBIAL METABOLITE 3-(4-HYDROXYPHENYL)-PROPIONIC ACID

Abstract Background Cranberries are a rich source of proanthocyanidins (PAC), a type of polyphenol with anti-cancer properties. We previously found that PAC, along with its microbially-derived metabolite 3-(4-hydroxyphenyl)-propionic acid (HPPA), trigger unique regulatory responses of microRNAs (miRNAs) in intestinal epithelial cells including the upregulation of miR-146a-5p. Both miR-146a-5p and miR363-3p are anti-inflammatory and downregulate anti-tumorigenic signalling pathways such as the interleukin (IL)-17 and wingless-related integration site (Wnt) respectively. miR-146a-5p also attenuates IL-17-promoting cytokines levels (e.g., IL-6). Aims To determine if miR-146a-5p and miR-363-3p respond to different physiologically relevant concentrations of PAC and HPPA in inflammatory and non-inflammatory conditions. Methods Fully differentiated Caco-2BBe1 colonic epithelial cells were treated with two doses of PAC-enriched cranberry extract (50μg/ml, 100μg/ml), HPPA (5μg/ml, 10μg/ml), or with Dulbecco’s Modified Eagle Medium (DMEM; control) for 24 hours, followed by IL-1β (1ng/ml) or mock stimulation for three hours. Human IL-6 homogeneous time-resolved fluorescence (HTRF) kits were used to quantify IL-6 in cell supernatant. RNA was extracted and used for miRNA profiling using Nanostring technology. Statistical analysis was performed in R version 4.2.1 with the R-packages NanostringDiff, NanostringNorm, and Pheatmap. Results At homeostasis, 42 and 2 (miR-146a and miR363-3p) miRNAs, respectively, uniquely responded to increasing PAC or HPPA concentrations. In the inflammatory state, no miRNAs responded to increasing concentrations of PAC and HPPA. However, the expression of miR-363-3p increased (qampersand:003C0.001) in response to 50μg/ml of PAC + IL-1β but decreased in response to 5μg/ml of HPPA + IL-1β , and the expression of miR-146a-5p increased (qampersand:003C0.001) in response to 5μg/ml of HPPA + IL-1β, and 50μg/ml PAC + IL-1β. Predicted miRNA gene-pathway analysis revealed that miR-146a-5p, miR-363-3p, and the other 42 miRNAs commonly target pathways involved in the tumorigenesis of colorectal cancer such as mitogen-activated protein kinases (MAPK), hedgehog, and Wnt pathways. Though, in inflamed cells, only HPPA (5 or 10 μg/ml) attenuated IL-6 secretion (pampersand:003C0.05), which may be driven by increased expression of miR-146a-5p. Conclusions These findings suggest that cranberries proanthocyanidins and their metabolites affect miRNAs involved in cancer related pathways in different manners and concentrations, which may depend on the inflammatory status. The gut microbiota may be partially responsible for unlocking these effects. Funding Agencies Ocean Spray Cranberries, Inc. and the Natural Sciences of Engineering Research Council of Canada (NSERC)

Bone marrow stem cells with or without superparamagnetic iron oxide nanoparticles as a magnetic targeting tool: Which is better in regeneration of neurolysed facial nerve? An experimental study

AimThis study was performed to evaluate neural regenerative capacities of bone marrow stem cells (BMSCs) with or without superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic targeting tool after neurolysis of the facial nerve (FN) in albino rats. MethodsThirty-eight male albino rats were selected. Two of them were euthanized for normal FN histology assessment. Thirty-six rats were injected with ethanol in the FN nerve for neurolysis induction and assessed one week post-operatively by eye blinking test. Animals were divided into three groups, each containing twelve rats: Group I (positive control) was injected with Dulbecco Modified Eagle's medium (DMEM-F12), group II was injected with BMSCs in DMEM-F12, and group III was injected with BMSCs in DMEM-F12 with poly l-lysine coated SPIONs (0.5 mmol/mL). Monitoring of SPIONs in the rat's body was carried out by MRI. A circular neodymium magnet N52 (0.57 T, 2 × 5 mm) was placed on each rat in group III just below the right ear at the site of surgery to attract SPIONs labeled BMSCs, left in place for 24 h, and then removed. From each group, six rats were euthanized at the end of the 4th and 8th week of treatment, respectively. The right FN trunks were extracted for routine histological examination using H&E stain. Immunohistochemical examination by anti-S100B was performed to characterize the thickness of the myelin sheath formed by the Schwann cells. Ultra-structural examination was performed to study changes in axons, myelin sheaths, and Schwann cells. ResultsRegeneration of nerve fibers, Schwan cells, and myelin sheaths was better in group II than in groups I and III histologically, immunohistochemically, and ultra-structurally. ConclusionBMSCs alone could ameliorate FN regeneration better than magnetic targeting treatment using BMSCs labeled with SPIONs.

Investigation the effect of Scrophularia striata hydroalcoholic Extract On Cell Death and Migration in Cervical Cancer Cell Line

Background: With the growing interest in plant-derived chemotherapeutic agents, there has been a significant rise in research exploring a broad range of plants in recent years. Scrophularia striata has gained attention due to its extensive medical applications. This study aimed to investigate the effect of S. striata extract on HeLa cervical cancer cells, specifically their migration, apoptosis, and necrosis. Methods: We first cultured HeLa cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. We then examined the cytotoxicity of S. striata extract at varying concentrations (0, 1, 10, 100, 500, and 1000 μg/mL) using the MTT assay after 24 hours. We evaluated the extent of wound healing using a scratch assay and analyzed the apoptosis activity of the extract using flow cytometry. Results: Our results showed that S. striata extract (IC50: 433.8 μg/mL) significantly enhances wound healing (P≤0.01) in cervical cancer and promotes apoptosis and necrosis of HeLa cells. Conclusion: Our findings suggest that S. striata may serve as an effective treatment for cervical cancer by inducing cell death and reducing migration.

The Key Role of Liraglutide in Preventing Autophagy of Vascular Smooth Muscle Cells in High Glucose Conditions

The glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LIRA) is a potential hypoglycemic drug with anti-atherosclerosis (AS) effects. Autophagy in the vascular smooth muscle cells (VSMCs) facilitates AS. However, the role of autophagy in the anti-AS mechanism of LIRA remains unclear. To examine the role and mechanisms of autophagy in LIRA’s improvement of the biological characteristics of VSMCs in high glucose conditions. Experimental animal study. VSMCs isolated from the thoracic aorta of male SD rats were subjected to a high glucose (HG) condition (25 mM) in Dulbecco’s Modified Eagle’s Medium with or without LIRA, the GLP-1 receptor antagonist exendin9-39 (Exe9-39), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and autophagy inhibitors (3-methyladenine [3-MA] and bafilomycin A1 [Baf A1]). Acridine orange staining, western blotting, transmission electron microscopy, and mCherry-GFP-LC3 transfection were performed to evaluate the autophagy flux. Additionally, VSMC migration, calcification, proliferation, and apoptosis in HG conditions were observed. Addition of LIRA alone or in combination with autophagy inhibitors significantly downregulated Beclin, increased the LC3-II/LC3-I ratio, and upregulated p62 in VSMCs in HG conditions. Furthermore, autophagolysosome formation was markedly curbed after treatment with LIRA and/or autophagy inhibitors. Inhibition of autophagy by LIRA and/or the autophagy inhibitors attenuated VSMC phenotype conversion, proliferation, migration, and calcification and promoted VSMC apoptosis in HG conditions. This protective role of LIRA was augmented by LY294002, but inhibited by Exe9-39. LIRA plays a significant role in the improvement of the biological features of VSMCs in HG conditions.

Culture Method and Regulation Mechanisms of Stages of C2C12 Cell Line Myogenesis

INTRODUCTION: C2C12 is an immortalized mouse myoblast cell line actively used as in vitro experimental models to study skeletal muscle metabolism in biomedical research. C2C12 cells can differentiate into myocytes under appropriate culture conditions. It is important to understand the specifics of each stage of myogenesis and its regulatory mechanisms for the targeted exposure and development of medical drugs, taking into account the mechanisms of pathogenesis of various diseases.
 AIM: To describe the method of culturing С2С12 cell line and to study regulatory mechanisms of С2С12 cell line myogenesis.
 MATERIALS AND METHODS: The study was conducted on C2C12 cell line. Differentiation of myoblasts was induced in the nutrient medium containing 2% horse serum. The differentiation stages were evaluated by studying the cells on the 1st, 4th and 7th days. Cells before differentiation were used as a comparison group. At each differentiation stage, the myoblast fusion index (IM) was evaluated with preliminary staining of the cells by Romanowsky–Giemsa method. The cell differentiation mechanism was evaluated by the level of myosin, a-actin, myogenic differentiation protein (MyoD), myogenin (MyoG) using Western blot method.
 RESULTS: On the 1st day of C2C12 cells culturing, formation of myotubes was observed (IM = 0.15 ± 0.05), on the 4th day — fusion of myoblasts with the formation of binucleated cells accompanied by an increase in the amount of MyoD and α-actin protein (IM = 0.44 ± 0.14). By the 7th day of differentiation, the fusion of cells increased with the formation of myotubes containing more than two nuclei; the content of MyoD and α-actin did not differ from the control, and the amount of MyoG and myosin increased (IM = 0.77 ± 0.04).
 CONCLUSION: The described method of culturing C2C12 cell line in Dulbecco’s Modified Eagle’s Medium with a high glucose content (4500 mg/l), containing 2% horse serum, L-glutamine (4 mМ), 100 Un/ml and 100 µg/ml of penicillin and streptomycin, is suitable for the formation of muscle cells, where MyoD and MyoG participate in the regulation of the increase in the amount of specific muscle proteins — α-actin and myosin.