Minimum Essential Medium Research Articles

Cytotoxic effect of Rotheca serrata on cancer cell lines MCF-7 and neuroblastoma SH-SY5Y.

Rotheca serrata (Lamiaceae), a highly medicinal plant is used as an antidote for snakebite and the plant possesses medicinal properties like hepatoprotective, antitussive, antioxidant, anticancer, neuro-protective, used in rheumatoid arthritis and is also a α-glucoside inhibitor. AIM OF THE STUDY: This work aimed to study the anticancerous effect of Rotheca serrata (root and leaf) on cancer cell lines MCF-7 (breast cancer cell line) and Neuroblastoma SH-SY5Y. This investigation was a preliminary one which supported the retrospective and safe use of plants as described in Ayurveda. Dulbecco's Modified Eagle Medium with High Glucose (DMEM-HG) for culturing MCF-7- Human Breast cancer cell line and Minimum essential Medium (MEM)+F12 medium for culturing SH-SY5Y- Homo sapiens bone marrow neuroblast were used. MTT assay measured the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The results indicated that the Methanolic extract of Rotheca serrata (root and leaf) showed high anticancer activity. Different concentrations of plant extracts (25, 50, 100, 200, 400 µg/ml) were used to study the anticancerous activity, amongst which the significant results were obtained for 400 µg/ml concentration (both root & leaf). Effective anticancer activity against MCF - 7 breast cancer cells was shown in methanoilc extracts and were expressed as IC 50 values; in root (IC 50 value = 61.8259 ± 7.428 µg/ml) and in leaf (IC 50 value = 78.1497 ± 6.316 µg/ml). The MTT assay in case of neuroblastoma (SH-SY5Y) cell lines revealed that 400 µg/ml concentration of leaf methanolic extract showed effective inhibition of cancer cells with IC 50 value 37.8462 ± 2.957 µg/ml as compared to IC 50 value of root methanolic extract which was 57.0895 ± 2.351 µg/ml. R. serrata possess anticancer activity against breast cancer cell line (MCF-7) and neuroblastoma (SH-SY 5Y) cell lines. This study may to design plant-based drugs without side effects. Dosage compensation for specific type of cancer needs to be monitored in patients with 1st stage.

Investigation of Ovarian and Non-ovarian Associated Factors Related to Follicular Population and Oocyte Maturation of Chadian Cattle Breeds

A cow can give birth to an average of 6-7 calves in her entire reproductive period. The remaining oocytes could be used for the in vitro production of embryos. The present study was conducted to evaluate the effects of ovarian and non-ovarian factors on the follicular population and oocyte maturation of three Chadian cattle breeds (Arab, Kouri, and Toupouri). For this purpose, the ovaries of 166 cycled cows were collected at the Farcha slaughterhouse of Chad and placed individually in labeled conical tubes containing 0.9% NaCl and 0.5 mg/ml penicillin-streptomycin. After clearing the ovaries of tissue debris, they were weighed, and the follicles were counted. The diameter of each follicle was measured and classified into three categories. A total of 2734 oocytes were collected in 28 days with a minimum of 97 per day by the slicing method using a 10X stereoscope. They were then classified into four groups according to the structure of their cumulus oophorus. Immature oocytes (class 1 and 2 [1455]) were placed in different culture media consisting of Minimum Essential Medium (MEM) alone, MEM with 10% follicular fluid, and MEM with 50% follicular fluid for oocyte maturation. The results indicated that the mean follicular population and mean oocyte yield were 24.71 ± 0.88 and 11.65 ± 0.94, respectively. The mean oocyte index and the number of cultivable oocytes for in vitro embryo production (class 1 and 2) were 1.03 ± 0.23 and 1.65 ± 0.94, respectively. The number of follicles observed in the age group of 6-9 years was higher than in other age groups. Oocyte yield was significantly higher in cows with a body condition score of 4-5 compared to average and lean cows. Among the different culture media used for oocyte maturation, the medium consisting of MEM plus 10% follicular fluid recorded a higher maturation rate than the other culture media. Cows aged 6-9 years had a higher maturation rate than other age groups. In conclusion, the good follicle (follicle that produced oocyte) and appropriate oocyte performance were observed in cows with body condition score 3-5 and an age range of 6-9 years.

Processing of Mg–Ca/tricalcium phosphate composites and their electrochemical properties in a cell culture medium

Biodegradable magnesium-based composites have recently attracted considerable attention for use in orthopedic implants on account of not only their biocompatibility but also the possibility of tuning their mechanical and corrosion properties. In this study, the effects of the fraction of tricalcium phosphate (TCP) and its crystal structure on the electrochemical properties in Eagle's minimal essential medium as well as the mechanical properties were examined using high-strength Mg–Ca/TCP composites with a fine-grained matrix (grain size: 1.2 µm) and relatively uniform TCP particle dispersion. The composites were prepared by extruding mixtures of the component powders at 540 K and exhibited high compressive yield strengths exceeding 290 MPa. Electrochemical tests revealed that the corrosion parameters were insensitive to both the fraction of TCP particles and their crystal structure. The fine-grained matrix may have overwhelmed the ability of the TCP dispersion to serve as a corrosion inhibitor. The chemical compositions of the cracked surface layer on the matrix and the white discontinuous corrosion products of the composites formed after the electrochemical tests were similar to those of the parent alloy.

In vitro culture and morphology of Neoechinorhynchus buttnerae (Eoacanthocephala: Neoechinorhynchidae) collected from the intestine of tambaqui (Colossoma macropomum) farmed in the Brazilian Amazon.

The tambaqui (Colossoma macropomum) is a species of great economic importance for fish farming in the Brazilian Amazon, and acanthocephaliasis caused by Neoechinorhynchus buttnerae (Golvan 1956) represents an obstacle to its production due to it causing severe morphological damage to the intestinal mucosa, thus impairing the absorption of nutrients and causing weight loss in the fish. Therefore, the establishment of in vitro protocols for evaluation of anthelmintic drugs is the first step to development of effective measures for in vivo control of this endoparasite. The present study evaluated the in vitro survival of N. buttnerae maintained in Eagle's minimum essential medium under different culture conditions. Three assays were carried out to evaluate whether temperature, supplementation with the antibiotics penicillin and streptomycin, and culture medium replacement or no replacement would influence the motility and morphology of the acanthocephalans. The results of the Kaplan-Meier analysis indicated that the use of culture in minimum essential medium together with penicillin and streptomycin prolonged the parasite's survival when kept at temperatures of 24 °C or 28 °C. We describe herein for first time an alternative protocol that is ideal for the in vitro culture of N. buttnerae. As such, this protocol ensures greater reliability in further in vitro studies with N. buttnerae.

Long-term Xenopus laevis tadpole -heart-organ-culture: Physiological changes in cholinergic and adrenergic sensitivities of tadpole heart with thyroxine-treatment

The present study clarified changes in physiological sensitivities of cultured Nieuwkoop and Faber stage 57 Xenopus laevis tadpole-organ-heart exposed to thyroxine (T4) using acetylcholine (ACh), norepinephrine (NE) and atropine. For preliminary life span and the chemical tests, 60% minimum essential medium (MEM), two types of modified Hank's balanced salt-solution-culture-media (MHBSS-CM) I and II containing relatively lower concentrations of amino acids and collagen were prepared. In preliminary lifespan-test of cultured tadpole hearts, the hearts maintained in 60% MEM was 50 days on average, whereas that of the tadpole-hearts in MHBSS-CMs was extended by 109 days on average, showing superior effectiveness of MHBSS-CMs. 4 min-stimulation by 5 × 10−9 M T4 tended to increase the tadpole heartbeat. 10−9 M ACh decreased the tadpole heartbeat. Frog-heart at 2–4 weeks after metamorphosis completion and tadpole heart treated with 5 × 10−10 M T4 for 45 h also responded to 10−9 M ACh, and low-resting hearts were restored to the control level with the competitive muscarinic antagonist 10−8 M atropine, whereas excessive exposure of 10−5 M atropine to T4-treated tadpole heart did not increase heartbeat in spite of the increased frog heartbeat over the control. 10−14 —10−12 M NE increase the tadpole heartbeat in a concentration-dependent manner, however, 10−12 M NE did not act to stimulate adrenergic receptors on both T4-treated tadpole- and the frog-hearts. These results suggest that T4 induces the desensitization of atropine-sensitive muscarinic and adrenergic receptors in organ-cultured tadpole-heart.

Comparison of media for a human peripheral blood mononuclear cell-based in vitro vaccine evaluation system.

Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

Interference-free quantitation of aromatic amino acids in two complex systems by three-way calibration with ultraviolet–visible spectrophotometer: Exploration of trilinear decomposition of spectrum-pH data

Aromatic amino acids play an extremely important role in life activities and participate in many biological processes. Their concentration levels are associated with a variety of diseases, such as phenylketonuria and colorectal cancer. Therefore, the quantification of aromatic amino acids is an important task. In the present work, a novel and rapid three-way analytical method was proposed to detect the levels of aromatic amino acids in prostate cancer cells (PC3 cells) and Dulbecco's modified minimal essential medium (DMEM cell culture), by using the affordable ultraviolet–visible spectrophotometer. First, spectrum-pH second-order data were designed per sample; Second, properties of the resulted spectrum-pH-sample three-way data were investigated by utilizing the parallel factor analysis (PARAFAC), alternating trilinear decomposition (ATLD), and constrained alternating trilinear decomposition (CATLD) algorithms, and a flexible scanning approach for determining the constraint parameters of CATLD was proposed; Third, a three-way calibration method based on the CATLD algorithm with the proposed scanning approach was developed for interference-free quantification of aromatic amino acids in these systems. The average relative predictive errors of validation (ARPEV) for phenylalanine, tyrosine, and tryptophan were 1.4%, 3.0%, and 0.7% in prostate cancer cells, and ARPEV for phenylalanine, tyrosine, and tryptophan were 4.1%, 1.2%, and 0.7% in DMEM cell culture. The predicted contents of tyrosine and tryptophan in DMEM cell culture were 64.2 ± 2.9 μg mL−1, 5.6 ± 0.3 μg mL−1, there are no significant differences in the concentrations between the developed analytical method and high performance liquid chromatography method. The proposed spectrum-pH-sample three-way calibration method based on CATLD algorithm can provide an interesting analytical strategy with high selectivity and accuracy for ultraviolet–visible spectrophotometer.

DETERMINATION OF CYTOTOXIC EFFECT OF AMYGDALIN IN DLD-1 CELL LINE AND ANTICYTOTOXIC EFFECT IN CCD-18CO CELL LINE

Objective: Amygdalin, which is part of the aromatic cyanogenic glycoside group, is found in plant seeds such as apricot, peach, plum, apple, pear, and cherry. It has been shown that amygdalin has anti-tumor properties against many cancers such as colon, breast, and lung cancer. This study aimed to determine the cytotoxic and anticytotoxic effects of amygdalin in human colon cancer cells (DLD-1) and normal colonic epithelium (CCD-18Co) using the MTT (3-(4,5-dimethylthiazol-2-YL)-2,5-diphenyltetrazolium bromide) test. 
 Materials and Methods: DLD-1 and CCD-18Co cells were grown in flasks containing Roswell Park Memorial Institute-1640 and Eagle's Minimum Essential Medium, respectively. Both cell groups were treated with amygdalin concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 mM for 24 hours. Then, 20% MTT dye was added to the wells of the aspirated plates and incubated for 3 hours. After the reaction was stopped with pure DMSO at the end of the period, the absorbance values of the plates were read spectrophotometrically at a wavelength of 570 nm.
 Results: The percent viability values for the DLD-1 cell line were found to be between 48.3-71.6% and the IC50 value was calculated as 74.03 mM. The viability values for the CCD-18Co cell line after the amygdalin treatment ranged from 101.6 to 117.9%.
 Conclusion: While amygdalin showed a cytotoxic effect in the DLD-1 cell line, it showed an anticytotoxic effect in the CCD-18Co cell line. In our study, it was determined that amygdalin decreased the viability of DLD-1 cancer cells in a dose-dependent manner and did not show cytotoxic effects on CCD18-Co normal epithelial cells. More comprehensive controlled clinical trials are needed to demonstrate the feasibility of using amygdalin in combination with other anti-tumor drugs and to develop the artificial synthesis of the active ingredients in amygdalin in order to increase the anti-tumor activities of these drugs.

Comparative Evaluation of Direct Smear and Culture Methods for Detection of Trichomonas gallinae Infection in Pigeon and Chicken of Assam

A study was carried out in order to compare the detection of Trichomonas gallinae by examination of smears of throat swab and culture of protozoa collected from both pigeon and chicken. In the present study, five culture media viz. Modified Diamond’s Media, Nutrient broth, Medium 199, Minimum Essential Medium (MEM) and Roswell Park Memorial Institute-1640 (RPMI-1640) were used for culture of T. gallinae. Microscopic examination of wet mount/ Giemsa stained smear and culture of the T. gallinae revealed the latter method to be superior to wet mount preparation or staining methods.

Identification of Potential Artefacts in In Vitro Measurement of Vanadium-Induced Reactive Oxygen Species (ROS) Production.

We investigated vanadium, i.e., a redox-active heavy metal widely known for the generation of oxidative stress in cultured mammalian cells, to determine its ability to interfere with common oxidative stress-related bioassays in cell-free conditions. We first assessed the prooxidant abilities (H2O2 level, oxidation of DHR 123, and DCFH-DA dyes) and antioxidant capacity (ABTS, RP, OH, and DPPH methods) of popular mammalian cell culture media, i.e., Minimal Essential Medium (MEM), Dulbecco's Minimal Essential Medium (DMEM), Dulbecco's Minimal Essential Medium-F12 (DMEM/F12), and RPMI 1640. Out of the four media studied, DMEM has the highest prooxidant and antioxidant properties, which is associated with the highest concentration of prooxidant and antioxidant nutrients in its formulation. The studied vanadium compounds, vanadyl sulphate (VOSO4), or sodium metavanadate (NaVO3) (100, 500, and 1000 µM), either slightly increased or decreased the level of H2O2 in the studied culture media. However, these changes were in the range of a few micromoles, and they should rather not interfere with the cytotoxic effect of vanadium on cells. However, the tested vanadium compounds significantly stimulated the oxidation of DCFH-DA and DHR123 in a cell-independent manner. The type of the culture media and their pro-oxidant and antioxidant abilities did not affect the intensity of oxidation of these dyes by vanadium, whereas the vanadium compound type was important, as VOSO4 stimulated DCFH-DA and DHR oxidation much more potently than NaVO3. Such interactions of vanadium with these probes may artefactually contribute to the oxidation of these dyes by reactive oxygen species induced by vanadium in cells.

Protective effect of teriparatide against vancomycin-induced cytotoxicity in osteoblasts

BackgroundIntrawound vancomycin powder is effective in preventing surgical site infection after spine surgery. In a previous study, vancomycin-induced cytotoxicity in osteoblasts was investigated in vitro, and vitamin D3 was verified to be a candidate drug aiding recovery from vancomycin-induced cytotoxicity. The treatment practices involving osteogenesis-promoting drugs vary widely. Teriparatide, an anabolic agent, highly promotes bone formation by inducing osteoblast activation, increasing bone formation and mineral density, and preventing vertebral fractures. Hence, teriparatide may be administered in combination with vancomycin. MethodsMC3T3-E1 cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator containing 5% CO2. The experimental concentrations of vancomycin (2500, 5000, and 7500 μg/mL) were determined based on previous reports and our preliminary experiments. Teriparatide (100 ng/mL) was administered concomitantly to prevent cytotoxicity in osteoblasts, using pulsed vancomycin for 24 h (measured at 1, 3, and 7 days). Cell numbers and morphological changes in cells treated with vancomycin or vancomycin plus 100 ng/mL teriparatide were measured. Osteoblast differentiation was assessed using alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red S staining. ResultsTeriparatide showed a recovery effect when vancomycin (7500 μg/mL) was administered only for 24 h. Microscopic examination revealed that teriparatide had a protective effect on osteoblasts exposed to 7500 μg/mL vancomycin. Addition of teriparatide led to the recovery of alkaline phosphatase staining and alizarin red staining. ConclusionVancomycin-induced cytotoxicity in osteoblasts could be inhibited by administering teriparatide concomitantly with vancomycin.

The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

The effects of cell culture conditions on premature hydrolysis of traceless ester-linked disulfide linkers

Disulfide acids are important for traceless release mechanisms in prodrugs and drug delivery applications. Their ability to self-immolate and release cargo due to environmental stimulus is invaluable. However, complex reactivity patterns may be overlooked as assays increase in complexity or are conducted in media of increasing biological relevance. Conclusions drawn from preliminary characterization in simple phosphate buffers are often applied to in vitro studies in which more complex media are used (e.g. containing glucose, amino acids, FBS, and the cell surface). We developed a model disulfide incorporating a fluorogenic dye as a reporter group in order to explore the generality of the disulfide prodrug system, and used this to explore the stability of disulfide esters in various contexts of increasing complexity. We found that our reporter molecules prematurely released cargo in a series of cell-containing and cell-free assays. We systematically reverse-engineered the components of a complex cell medium and found that FBS was capable of interfering with disulfide-based prodrug linkers, triggering the release of conjugated 4-methylumbelliferone (4-MU) from representative reporter molecules. FBS consistently induced 4-MU release in complete media (i.e. DMEM and RPMI 1640), minimal essential media, and pure water. Signs of 4-MU release were mitigated when FBS was subjected to intense heat (>100 °C) or esterase-specific protease inhibitor cocktail (PIC), indicating that esterases from the serum were capable of triggering cargo release using a hydrolysis mechanism that is separate from the desired reductive cleavage pathway. These findings are important because they show that variance in models may hide unexpected results, which calls for more meticulous consideration of control experiments when developing stimulus-release agents for biological applications.

Antitumor Effects of L-citrulline on Hela Cervical Cancer Cell Lines.

Cervical cancer is the deadliest gynecological malignancy. This study aims to examine the anticancer effects of L-citrulline on HeLa cell culture. HeLa cells were cultured in complete Eagle's minimum essential medium. HeLa cells were seeded in 96-well plates and incubated with L-citrulline. After incubation, MTT dye was added and incubated. Annexin- V technique was used to test the apoptosis. The activated caspases of HeLa cells by L-citrulline exposure were measured with the Caspase 3/7 technique. One-way variance analysis was conducted for statistical analysis by using GraphPad Prism 6.0 for Windows. L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner in application times of 24 and 48 hours. The IC50 dose of L-citrulline was 0.19 and 0.16 mg/mL at 24 and 48 hours, respectively. When HeLa cells were exposed to an IC50 dose of L-citrulline for 24 hours, the percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.75%, 23.05%, and 12.75%, respectively. The differences in the wideness of the scratch area were observed at the initial stage and after 24 hours of applying L-citrulline. L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner. Based on Annexin and Caspase findings, it can be concluded that L-citrulline exerted a pro-apoptotic effect on HeLa cells in only a short exposure time. L-citrulline also showed a migration inhibitory effect. The findings of this study indicate L-citrulline to be worthy of investigation for its anticancer activities in vitro and in vivo, and as a candidate for cancer therapy.

Comparative study of five culture media for in vitro culture of Trichomonas gallinae from birds in Assam

A study was carried out in order to compare the in vitro efficacy of five different culture media against the flagellate protozoa, Trichomonas gallinae. For in vitro culture, oral swabs of T. gallinae were collected randomly from oropharynx and crop after visual inspection of birds as pigeon, duck, chicken and quail with a sterilized cotton swab and cultivated in five media namely modified Diamond media, Medium199, Minimum Essential Medium (MEM), RPMI 1640 and Nutrient broth. The initial inoculation of trophozoites into each media was 1X103cell/ml. Among all the five media, Medium 199 showed the highest growth rate and motility of the organisms till 7 days after initial inoculation. All media were found suitable for harvesting and sub culture of T. gallinae under in vitro condition.

Effect of endometrial cell-conditioned medium and platelet-rich plasma on the developmental competence of mouse preantral follicles: An in vitro study.

ObjectiveThe aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization.MethodsIn total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction.ResultsIn the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups.ConclusionOverall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

In-vitro corrosion behaviors of extruded Mg–Ga alloys in alpha minimum essential medium

The in-vitro corrosion behaviors of extruded Mg–xGa (x = 1, 3, 5) alloys in alpha minimum essential medium (α-MEM) were investigated. The bilayer corrosion film was composed of an outer layer dominated by amorphous phosphates–carbonates and an inner layer dominated by MgO–Mg(OH)2. Pure Mg and Mg–1Ga exhibited low corrosion rates and homogeneous corrosion behaviors owing to the protection from the compact corrosion films. By contrast, Mg–3Ga and Mg–5Ga exhibited high corrosion rates and inhomogeneous corrosion behaviors owing to the bulky micro-galvanic corrosion caused by Mg5Ga2. Based on these results, possible corrosion models were developed.

Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles.

This study characterized the expression of melatonin receptor type 1 (MT1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α-modified minimum essential mediumalone (α-MEM+ ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3, Akt, phosphorylated-Akt, and phosphorylated-FOXO3a(p-FOXO3a). The immunohistochemical localization of the MT1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α-MEM+ and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase-3-positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p-FOXO3a. In conclusion, MT1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.

Osteoimmunomodulatory potential of 3D printed submicron patterns assessed in a direct co-culture model.

Modulation of the immune response following the implantation of biomaterials can have beneficial effects on bone regeneration. This involves complex interactions between the inflammatory and osteogenic cells. Therefore, the study of cell-cell interactions using direct co-culture models integrated with biomaterials is of great interest. This research aimed to study the viability, morphology, and osteogenic activity of preosteoblasts (OBs) co-cultured with pro-inflammatory macrophages (M1s) on the 3D printed (non)patterned surfaces. OBs and M1s remained alive and proliferated actively for 14days in the mixture of Dulbecco's Modified Eagle's Medium (DMEM) and alpha Minimum Essential Medium (α-MEM) (1:1), regardless of the cell ratio in the co-cultures. The spatial organization of the two types of cells changed with the time of culture from an initially uniform cell distribution to the formation of a thick layer of OBs covered by clusters of M1s. On day 7, the expression of PGE2 and TNF-α were upregulated in the co-culture relative to the mono-culture of OBs and M1s. The inflammation decreased differentiation and matrix mineralization of OBs after 28days of culture. Interestingly, the incorporation of 3D printed submicron pillars into the direct co-culture model enhanced the differentiation of preosteoblasts, as shown by relatively higher RUNX2 expression, thereby revealing the osteoimmunomodulatory potential of such surface patterns.

High D-glucose levels induce ACE2 expression via GLUT1 in human airway epithelial cell line Calu-3

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2) receptors. ACE2 is expressed on human airway epithelial cells. Increased ACE2 expression may be associated with potentially high risk of COVID-19. However, the factors responsible for the regulation of ACE2 expression in human airway epithelial cells are unknown. Furthermore, hyperglycemia is a risk factor for poor disease prognosis.ResultsIn this study, we investigated the effects of D-glucose on ACE2 mRNA and protein expressions in Calu-3 bronchial submucosal cells. The cells were cultured in minimal essential medium containing different D-glucose concentrations. After 48 and 72 h of high D-glucose (1000 mg/dL) treatment, ACE2 mRNA expressions were significantly increased. ACE2 protein expressions were significantly increased after 24 h of high D-glucose treatment. ACE2 mRNA expression was enhanced by a D-glucose concentration of 550 mg/dL or more after 72 h of treatment. In addition, we investigated the role of glucose transporters (GLUTs) in Calu-3 cells. ACE2 mRNA and protein expressions were suppressed by the GLUT1 inhibitor BAY-876 in high D-glucose-treated Calu-3 cells. GLUT-1 siRNA was also used and ACE2 mRNA expressions were suppressed in high D-glucose-treated Calu-3 cells with GLUT-1 knockdown.ConclusionsThis is the first report indicating that high D-glucose levels induced ACE2 expression via GLUT1 in bronchial submucosal cells in vitro. As hyperglycemia can be treated appropriately, these findings could help reduce the risk of worsening of coronavirus disease 2019.