MEM Medium Research Articles

Establishment of Angora rabbits’ whisker hair follicle model and optimal culture conditions

To establish the model of whisker hair follicle culture in vitro and explore the best culture conditions, the whisker hair follicles of Angora rabbits were separated with stereomicroscope and cultured in William’s E, DMEM, MEM media. The surface of the cultured whisker hair follicles was not damaged due to manual operation, resulting in the hair shaft’s growth. This indicated the success of the in vitro whisker hair follicle model. The hair shaft grew at the fastest rate in the William’s E culture (p < 0.05), which was significantly higher than that in the DMEM and MEM media. The hematoxylin-eosin results showed that compared to the William’s E group, the atrophy of whisker hair follicles in the DMEM and MEM media was evident, especially in the MEM medium. PCNA immunofluorescence staining was employed to detect the expression of whisker hair follicles. The results showed that the PCNA positive expression of the William’s E group was significantly stronger than that of the DMEM and MEM groups. Furthermore, CCK-8 and Annexin V-FITC/PI methods were used to detect the proliferation and apoptosis of the dermal papilla cells (DPCs). The results of this study provide a model for studying the hair growth of fur animals.

Ketahanan Eksplan Embrio Ayam Dalam Media In Vitro

Science has implemented the cell cultures as the primary for research such as a model system, toxicity tests, cancer, viruses, cell-based industries, genetic counseling, genetic engineering, gene therapy, drug screening and the latest is invitro meat. The purpose of this study was to determine the resistance of chicken embryo eksplan cells in invitro media. The research design was in the form of laboratory experiments. Explants taken 4 day old chick embryos, which were cultured in MEM media + serum. The results showed fibroblast cell cultures which survive 2 days. The most influential factor is the environment that is less supportive of the growth of explant chicken embryo cell

Synthesis and Biological Profiles of Some Benzimidazolyl-chalcones as Anti-leishmanial and Trypanocidal Agents

Background: Benzimidazole constitutes a starting point for the development of new antiprotozoal agents since this nucleus exists in several pharmacologically significant molecules, in particular possessing antifungal, antiviral, antibacterial, and antiparasitic properties.&#x0D; Objective: The present study aimed to identify a molecular hit likely to be developed as an anti-leishmanial and antitrypanosomal drug candidate.&#x0D; Methods: Thus, 12 hybrids of chalcone or benzimidazolyl-arylpropenones were synthesized and screened in vitro for anti-leishmanial activity against promastigotes of Leishmania donovani. The microculture tetrazolium assay was used to determine their potential to inhibit 50% of a parasite growth (IC50).&#x0D; Results: Two compounds among 5-chlorobenzimidazole-chalcones (4a and 4c), which exhibited potent activity (IC50&lt;1 μM) against L. donovani and one derivative (4d) poorly effective against L. donovani (IC50&gt;50 μM) were selected to check their trypanocidal activity. Lethal concentration (LC100) values of these compounds were estimated by using observations on the viability of trypomastigotes of Trypanosoma brucei brucei in MEM medium with Earle’s salts and L-glutamine. Seven of the tested compounds (4a, 4b, 4c, 4e, 4g, 4h, and 4j) showed particularly higher inhibitory activity than pentamidine (IC50= 7.6 μM) against L. donovani promastigotes with IC50 values in a range from 0.5 to 1.8 μM. In addition, the 2’-chlorine derivative (4c), displayed potent anti-trypanosomal activity comparable to those of melarsoprol, used as reference drug.&#x0D; Conclusion: These results support this series of benzimidazole holding arylpropenone group in position 2 as a starting point for future optimization to get novel agents active against leishmaniasis and trypanosomiasis.

Effect of secondary metabolites of Berberis vulgaris and Petasites hybridus on apoptosis and cell cycle of HeLa cell line.

e15063 Background: The study of the possible antitumor activity of secondary plant metabolites either in the form of individual agents or in combination with clinically used drugs is considered as a promising direction in the therapy of malignant tumors. The aim of this study was an estimation of antitumor activity of secondary plant metabolites in the in vitro experiment on the HeLa cell line. Methods: Secondary metabolites were extracted from plant raw materials and were isolated by preparative chromatography. Determination of their composition was carried out using HPLC; obtained compound structures were identified by NMR. We selected 4 secondary metabolites from Petasites hybridus for testing: (2,4-dihydroxy-2,5-dimethylfuran-3 (2H), 5-(hydroxymethyl) furan-2-carbaldehyde, 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, Corynan), and 1 secondary metabolite from Berberis vulgaris : a berberine chloride (BBR, C20H18NO4+Cl- is a derivative of 5,6-dihydrobenzo[a,g] isoquinolinium). HeLa CCL2 cultivation was carried out under standard conditions in the MEM medium. When reaching the 75-80% confluence level we replaced nutrient medium with the introduction of secondary metabolites (concentration 4 and 12 μg/ml) and cultured for 24 and 72 hours. Cell survival was determined on a NanoEnTek JuliFl counter (Korea) in the presence of 0.4% trypan blue. Apoptosis was assessed on a flow cytometer BD FACSCanto II using FITC Annexin V Apoptosis Detection Kit I. Post-exposure copy number and expression were assessed by Real-time PCR with a panel of genes CASP9, CASP8, CASP3, TP53, MDM2, BAX, BCL2, CDK1, BRCA1, BRCA 2, RB1. Results: All obtained data were normalized by negative control. When we used 4 μg/ml berberine solution with 72-hour exposition, the proapoptogenic effect was maximal, causing the death of 67.2% of HeLa cells (29.3% early apoptosis, 37.2% late apoptosis, 0.7% necrosis). Within 24 hours, berberine at the same concentration caused a 2-fold increase in TP53 expression relative to MDM2. An increase in its concentration to 12 μg/ml and exposure for up to 72 hours led to a 31-fold increase in TP53/MDM2. The terpenoid 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde at a concentration of 12 μg/ml after 72 hours of cultivation caused a 6.5-fold increase in the TP53/MDM2 ratio. The corynan alkaloid (12 μg/ml) at an exposure of 72 hours increased the BAX/BCL ratio by 2.4 times. There were no statistically significant differences in the expression and copy number of the remaining genes studied. Conclusions: Berberine, corynan, 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde showed high promise in the HeLa cell line in vitro, since they surpassed the antitumor activity of other metabolites of Berberis vulgaris and Petasites hybridus.

Ovarian toxicity by fusariotoxins in pigs: Does it imply in oxidative stress?

Mycotoxins are natural contaminants of food and feed occurring worldwide. Deoxynivalenol (DON) and fumonisin B1 (FB1) are the most frequent fusariotoxins and induce immune and intestinal toxicity in humans and animals. Recently, an association between mycotoxins exposure and impaired fertility has been suggested. However, the effects of these mycotoxins on the reproductive system are not well established. This study aimed to evaluate the effects of FB1 and DON, in combination or alone, on the ovarian morphology and oxidative responses using porcine explants. Seventy-two explants were obtained from six pigs and submitted to the following treatments: control (MEM medium), DON (10 μM), FB1 (100 μM FB1), and DON + FB1 (10 μM + 100 μM). Histological and immunohistochemical assays were performed to evaluate ovarian changes, cell proliferation, and apoptosis. Oxidative stress response was evaluated through lipid peroxidation and antioxidant capacity response assays. The exposure to mycotoxins induced significant histological changes in the ovaries, which were characterized by a decrease in viable follicles and increase in degenerated follicles. A significant decrease in granulosa cell proliferation was observed in explants exposed to all mycotoxins. In addition the multi-contaminated treatment was responsible for an increase in the cell apoptosis index of growing follicles. On the other hand, the FB1 and multi-contaminated treatments induced a significant decrease in lipid peroxidation accompanied by an increase in antioxidant responses. Altogether, our results indicate a reproductive toxicity induced by fusariotoxins. Moreover, mycotoxins, alone or in combination, modulate oxidative stress response, interfering with the production of free radicals and affecting the reproductive capacity of pigs.

Bone marrow mesenchymal cells: polymorphism associated with transformation of rough endoplasmic reticulum.

To understand the behavior and function of bone-marrow mesenchymal cells (BMMCs), we overviewed the morphological presentation of BMMCs in bone-marrow granules (b-BMMCs), isolated BMMCs (i-BMMCs), and BMMCs (c-BMMCs) cultured in H4434 methylcellulose semisolid and MEM media. All samples were derived from bone-marrow aspirates of 30 patients with hematocytopenia. Light microscopy exhibited b-BMMCs and i-BMMCs characterized by abundant cytoplasm and irregular shape in bone-marrow smears, as well as c-BMMCs in culture conditions. Scanning electron microscopy demonstrated cultured c-BMMCs with a sheet-like feature enveloping hematopoietic cells. Transmission electron microscopy revealed b-BMMCs constructing a honeycomb-like structure by thin bifurcate processes among hematopoietic cells. Furthermore, i-BMMCs had bifurcate parapodiums on the surface and prominent rough endoplasmic reticulum (rER) connected with the plasmalemma of the parapodiums. The detailed images suggested that rER may serve as a membrane resource for plasmalemmal expansion in BMMCs in bone marrow.

Osteogenic Effects of Naringenin Are Mediated Through the Activation of AMPK Pathway

Abstract Objectives Bone marrow mesenchymal stem cells are the precursor cells for adipocytes and osteoblasts. The accumulation of adipocytes in bone marrow at the expense of osteoblastogenesis is a major factor contributing to age-related bone loss. Grapefruit flavonoid naringenin (NAR) was shown to promote osteogenesis but the mechanism behind these effects is not clear. In this study, we propose to demonstrate that the osteogenic effects of NAR are mediated through the activation of adenosine monophosphate-activated kinase (AMPK) pathway. Methods MC3T3-E1 pre-osteoblast cells were cultured using alpha MEM medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Osteogenic differentiation was induced by using differentiation medium purchased from Stem Cell Technologies. Western blotting, ELISA and alizarin red assays were conducted to test our hypothesis. Results NAR increased calcium deposition in MC3T3 cells compared to DMSO control after 21-day differentiation period. Additionally, in the presence of AMPK inhibitor Ara-A, NAR-induced calcium deposition was suppressed compared to NAR alone. Furthermore, while NAR increased phosphorylation of AMPK, the combination of NAR and Ara-A significantly decreased AMPK phosphorylation. Finally, Ara-A suppressed NAR-induced expression of osteogenic markers including Runx2, osteoprotegerin and osteocalcin suggesting that NAR-induced osteogenesis is partly mediated through AMPK pathway. All data were expressed as the mean ± SEM, minimum n = 3. Comparisons were made by using one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. Conclusions The suppression of osteogenic markers and calcium deposition in the presence of NAR plus Ara-A, an AMPK inhibitor, compared to NAR alone provide evidence that the observed osteogenic effects of NAR are in part mediated through the activation of AMPK pathway. These data combined with our previous reports of anti-adipogenic effects of NAR suggest that NAR may have potential therapeutic applications for osteoporosis by not only promoting osteogenesis but also inhibiting adipogenesis in the bone marrow. Funding Sources Division of Research, PCOM and NIH-1R03 AI128254–01A1.

Fructose-Induced Crabtree Effect: Inhibition of Respiration and Stimulation of Glycolysis in HepG2 Cells

Background: Non-alcoholic fatty liver disease (NAFLD) is approaching epidemic proportions in the US. Increased consumption of fructose (in the form of sucrose or high-fructose corn syrup) has been implicated in the development of NAFLD. The acute effects of fructose on energy metabolism of the liver have not been explored yet. Glucose is known to shift the energy source of ATP generation from respiration to glycolysis (the Crabtree effect). The liver is unique since glucose metabolism is very low in normoglycemia, however, fructose is readily metabolized and is not subject to enzyme regulation that limits the metabolism of glucose in liver. We hypothesized that fructose could induce the Crabtree effect in liver cells. Methods: The hepatoma cell line (HepG2) was cultured in MEM medium. Energy metabolism was investigated using the Agilent Seahorse XFe24 Analyzer, which simultaneously measures respiration (as oxygen consumption rate, OCR) and glycolysis (related to extracellular acidification rate, ECAR). These rates were used to estimate ATP production rates (JATP; see Mookerjee et al., 2018, J.Biol..Chem. 292:7189). Results: Glucose (10 mM) inhibited mitochondrial (oxidative) ATP production by 67% and, at the same time, increased glycolytic ATP production 6.2-fold. The effects of fructose were similar albeit somewhat smaller: fructose (10 mM) inhibited mitochondrial ATP production by 48% and increased glycolytic ATP production 3.6-fold. The effects of glucose and fructose were not additive. Conclusions: This preliminary study shows that fructose alters energy metabolism in a model system of the liver. A similar effect in may contribute to the development of NAFLD.

P11.14 The addition of hyperbaric oxygen to the treatment of T98G glioblastoma cell cultures with selected CK2 inhibitor TDB enhances its anti-tumour effect

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive glial neoplasm characterized by infiltrative cell growth in adults. The current standard of care for high-grade glioma patients includes surgery, radiotherapy and chemotherapy using temozolomide and other alkylating agents. Despite this, median survival of the patients with GBM is less than two years. One of the main factors related to the progression of neoplastic cells is hyperactivity of protein kinases, including CK2 kinase and a high level of tumor hypoxia, both of which also contribute to tumour resistance to treatment. Our previous study indicated that some CK2 inhibitors exhibit anti-proliferative effect on glioma cells. The aim of this study was to examine whether HBO may enhance the anti-tumour efficacy of 1-(β-D-2’-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) - CK2 inhibitor, under combination HBO/TDB therapy. MATERIAL AND METHODS Studies were performed on human glioblastoma T98G cell line (ATCC). Cells were cultured in MEM medium supplemented with 1-(β-D-2’-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole in concentrations of 0.1 μM; 1 μM, 25 μM, 50 μM, 75μM, 100 μM. Cultures were sustained in various oxygen conditions: normoxia for 24 and 48 hours or1 hour of hyperbaric oxygen (HBO, 97,5% O2/2,5% CO2, pressure of 2 ATA) followed by 23 hours of normoxia (21% O2/5% CO2/78% N2). Control groups included the cultures sustained in normoxia conditions. The viability of glioma cell lines was established at 24 and 48 hours post TDB treatment by Cell Titer 96®A Queous One Solution Cell Proliferation Assay. RESULTS The study evidenced that TDB inhibited the viability of neoplastic cells at a concentration of 75 μM after 24 and 48 hours. Administration of hyperbaric oxygen alone also caused a reduction in viability of glioblastoma cells. The cytotoxic effect of the tested CK2 inhibitor was significantly enhanced when tested compound was combined with HBO. What’s more, under the influence of hyperbaric oxygen, TDB significantly reduced the viability of tumor cells already at a concentration of 25 μM after 24 hours. CONCLUSIONS Hyperbaric oxygen administration significantly reduces the viability of T98G line cells and increases their sensitivity to the tested CK2 kinase inhibitor. The beneficial cytotoxic effect of TDB/HBO can be obtained at lower concentrations of the tested compound and after shorter exposure time as compared to its administration without HBO. Acknowledgement: The research was supported by the Foundation for the Development of Diagnostic and Therapy, Warsaw

Study on safflower yellow for injection based on cell degranulation and acute anaphylactoid model

This paper was aimed to establish screening methods of anaphylactoid reaction caused by safflower yellow for injection based on RBL-2 H3 cell degranulation model and mice model for acute anaphylactoid reaction,and evaluate the hypersensitivity caused by safflower yellow for injection from different batches. An in vitro cell model was used to keep the cells stimulated for an hour with different batches of safflower yellow for injection as the drug group,serum-free MEM medium as negative control group and 30 mg·L-1 C48/80 as positive control group respectively. The supernatant was then absorbed,and neutral red staining technique was used to detect the effect of safflower yellow injection on the degranulation of RBL-2 H3 cells with the positive cell rate of degranulation as the indicator.An in vivo model was established to validate the experimental results,and mice model for acute anaphylactoid reaction and ELISA method were adopted to detect the plasma histamine content,and screen the hypersensitivity caused by safflower yellow for injection at the animal level by using plasma histamine content as a test index. The results of the neutral red staining experiments showed that the positive control C48/80 could cause cell degranulation,and most of the cells were deeply stained. There was significant difference in positive cell rate between different batches of safflower yellow and positive control group. In the mice model for acute anaphylactoid reaction,it was found that the positive control C48/80 significantly increased the histamine content in the plasma of mice,while the safflower yellow in each batch did not cause a significant increase in plasma histamine( P<0. 000 1). The mechanism of anaphylactoid reaction is relatively complicated. This study was mainly based on the release of histamine and other active substances by degranulation of mast cells. No significant degranulation reaction of RBL-2 H3 cells induced by safflower yellow for injection was detected,nor was the plasma histamine level significantly increased in mice from the in vitro and in vivo aspects.

IN VITRO EVALUATION OF ANTI-CANCER POTENTIAL OF A3 ADENOSINE RECEPTOR AGONIST ON A549 HUMAN LUNG CANCER CELL LINE

Objective: The objective of this study was to evaluate the cytotoxic potential of A3AR agonist (ABMECA) against human lung cancer cell line A549 by using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.&#x0D; Methods: Adenocarcinoma cell line A549 was used to assess MTT based cells viability. In vitro cytotoxic activity was evaluated for 3 different concentration of doxorubicin and A3AR by MTT cytotoxicity assay. Cytotoxicity assay carried out for 3 consecutive days that involves culturing cells into Dulbecco’s MEM medium modified with 10% FBS for 24 h then treatment with different dose of standard and test drug with incubation period of 24 h followed by treatment with MTT for estimation of cytotoxicity and finally, optical density (OD) was measured at 570-630 nm.&#x0D; Results: Different concentration of doxorubicin (1, 5, 10 µM) and ABMECA (10-6M, 10-5M and 10-4M) shown dose-dependent cytotoxicity. There was a significant decrease (p&lt;0.05) in cell viability in both doxorubicin and ABMECA concentration in a dose-dependent manner. This study may guide further for in vivo evaluation of test drug in the lung cancer model.&#x0D; Conclusion: A3 Adenosine Receptor agonist could be potential moiety for the treatment of lung cancer and it would require in vivo study for further research.

P04.48 Role of the expression of protein kinase D1 and its phosphorylated forms in glioma-derived cell line exposed to modified isothiourea derivative ZKK-3 combined with hyperbaric oxygen therapy

Glioblastoma (GBM) is the most malignant primary tumour of the central nervous system. Its standard treatment includes surgery, radiotherapy and chemotherapy using temozolomide. Despite this, therapies still seems to be insufficient and median survival of the patients with GBM is less than 16 months. One of the main factors involved in glioma progression is the high level of tumour hypoxia, which contributes also to its resistance to treatment. In our previous studies we demonstrated that combination of hyperbaric oxygenation with novel pentabromobenzylisothiourea ZKK-3 resulted in an improvement of GBM cells oxygenation status and the reduction of their proliferation and viability in vitro. There is a high structural similarity between isothioureas and known casein kinase 2 (CK2) inhibitors. Nevertheless, ZKK-3 does not suppress CK2 expression, while it inhibits other protein kinases, including protein kinase D1 (PKD1), which promotes tumour growth and mediates detoxification of mitochondrial reactive oxygen species. The aim of this study was to assess the changes of the expression of PKD1 and its phosphorylated forms - pPKD1 (Ser 916) and pPKD1 (Ser 744/748), in glioma-derived cells treated with ZKK-3 under various oxygen conditions in vitro. Studies were performed on human glioblastoma T98G cell line (ATCC). Cells were cultured in MEM medium supplemented with N,N’-dimethyl-S-(2,3,4,5,6-pentabromobenzyl)-isothiouronium bromide (ZKK-3) in concentrations of 10 μM, 25 μM and 50 μM. Cultures were sustained in various oxygen conditions: normoxia, hypoxia, HBO, double hypoxia, hypoxia/HBO for 24 hour and then lysed with RIPA Lysis Buffer System (Santa Cruz). Using Western Blot protocol and appropriate primary and secondary antibodies, levels of tested protein kinases were determined. As the loading control β-actin was used. Administration of ZKK-3 did not affect PKD1 expression in glioblastoma cell line. Nevertheless, treatment with 50 µM of selected agent resulted in significant changes in the level of phosphorylated form of tested protein: diminution of pPKD1 (Ser 916) level and elevation of pPKD1 (Ser 744/748) level. Exposition of T98G cells to various oxygen conditions only slightly altered the expression of PKD1 and its phosphorylated forms in comparison to standard conditions, in both control and ZKK-3-treated groups, but observed differences were generally statistically insignificant. Obtained results suggested that PKD1-inhibiting properties of ZKK-3 were related with blocking the phosphorylation of that kinase at Ser 916. Unchanged expression of PKD1 as well as its phosphorylated forms under the influence of different oxygen conditions allowed to exclude PKD1 as a key protein for antitumour properties of ZKK-3/HBO combination strategy. The research was supported by KNOW-MMRC project.

A new classification of the germinal vesicle chromatin configurations in pig oocytes.

Reported classifications of germinal vesicle (GV) chromatin configurations in pig oocytes were not done by uniform standards and they were not well correlated with oocyte competence. In this study, GV chromatin of pig oocytes was classified into nonsurrounded nucleolus (NSN), surrounded nucleolus (SN), partly NSN (pNSN) and SN (pSN), prematurely condensed NSN (cNSN), pNSN (cpNSN) and pSN (cpSN), and early diakinesis (ED) patterns. During in vitro maturation in 199 medium, NSN oocytes from 1 to 2 mm follicles went consecutively through pNSN, pSN, cpSN, and ED before undergoing GV breakdown, and chromatin in some SN oocytes from 3 to 6 mm follicles re-decondensed into a re-decondensation (RDC) configuration. Under unfavorable conditions such as follicle atresia, ovary handling or maturation in simple MEM medium, however, premature chromatin condensation occurred, forming cNSN, cpNSN, and cpSN patterns. While all NSN and pNSN and some pSN and RDC oocytes actively transcribed, no cNSN, cpNSN, or cpSN oocytes showed transcription. Maturation and embryo culture suggested that SN and pSN oocytes were more competent than NSN and pNSN oocytes; cpSN oocytes were more competent than cNSN/cpNSN oocytes; and only RDC oocytes could develop into blastocysts. It is concluded that the newly classified chromatin configurations are more closely correlated with oocyte competence than those reported previously.

Effects of Bone Marrow Stromal Cell Transplantation on Repair of Bone Defect in Rats

Background: Mesenchymal stem cells are one type of adult stem cells, which are able to give rise to mesodermal origin tissues. The application of mesenchymal stem cells for tissue and organ regeneration offer advantages because of the relative ease of collection and their potential to differentiate to 3 cell lineages. Objectives: This research was designed to study and evaluate the effect of mesenchymal stem cells on the repair of bone defects in Wistar rat models and to compare autologous and non-autologous cell transplantation in repairing bone defects. Methods: The mesenchymal stem cells were cultured and expanded in MEM medium supplemented with 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin by incubation at 37°C in 5% CO2. Immunocytochemistry analysis was performed using CD44 and fibronectin markers to detect the mesenchymal stem cells. A transcortical defect was created within the distal epiphyses of the femur bone. After 3 days of injury, Brdu-labeled cells were injected at the site of injury. The animals were sacrificed after 4 weeks of transplantation and the femurs with the recipient area were removed and the length, weight, area, density, and biomechanical parameters were evaluated. Results: The obtained results showed that although neither non-autologous nor autologous cell transplantation significantly improved the effect on length and area of the defective femur bone, the mass and bone density of the cell-injected transplanted groups had a significant increase in comparison with the control and sham groups. Conclusion: It seems that cell transplantation could improve these parameters, improving the mean mass, length, area, and density of parameters during the the regeneration process.

Chitosan nanoencapsulation of flavonoids enhances their quorum sensing and biofilm formation inhibitory activities against an E.coli Top 10 biosensor.

Phytochemicals have been found to be promising alternatives to conventional antibiotic therapies for the control of bacterial infections, as they may entail less selective pressure and hence reduce the development of resistance. This study involved examining the inhibition of biofilm formation and of quorum sensing (QS), and the cytotoxicity on mammalian cells of two flavonoids, quercetin and baicalein, in free form and associated into chitosan-based nanocapsules. This was done by use of a transformed E. coli Top 10 biosensor strain, while the cytotoxicity was evaluated on MDCK-C7 cells. In free form, application both flavonoids exhibited slight inhibitory activity on the QS response and biofilm formation, a scenario that was improved positively upon encapsulation with chitosan (Mw ∼115,000 g/mol and DA ∼42%). The association efficiency of 99% (quercetin) and 87% (baicalein) was determined, and each formulation had an average diameter of 190 ± 4 and 187 ± 2 nm, and zeta (ζ) potential of +48.1 ± 2.03 and +48.4 ± 3.46 mV, respectively. Both types of systems were stable against aggregation in M9 and MEM media. The in vitro release kinetics data of both flavonoids seemed to be similar with only ∼20% released over the first 5 h, or ∼10% over the first 4 h, respectively, with subsequent sudden release increase up to ∼40% in both cases. The free phytochemicals seemed to be cytotoxic to MDCK-C7 cells at higher doses, however, upon nanoencapsulation, a cytoprotective effect was evidenced. We have gained proof-of-principle of the advantages of encapsulation of two bioactive flavonoids.

Efficacy of a repeat dose injection of CLR 131 (I-131-CLR1404) in U87-MG tumor-bearing athymic nude mice.

e14089 Background: CLR 131 is a novel radioiodinated therapeutic that exploits the selective uptake and retention of phospholipid ethers (PLEs) by malignant cells. Study was to evaluate the therapeutic effect of CLR 131 when administered as a repeat dose intravenous injection in U87-MG tumor bearing mice. Malignant gliomas are associated with a low survival rate, and treatment with chemotherapy, external beam radiation, or surgery have shown limited effectiveness. In this study, therapeutic effect was evaluated by measurement of tumor volume increase and by survival of CLR 131 treated animals compared to animals dosed with a I-127-CLR1404 control. Methods: The U87-MG cell line (human glioma) was purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in MEM media supplemented with 10% fetal bovine serum. Twenty female athymic nude mice (Harlan, Indianapolis, IN) weighing between 16-20 g were anaesthetized with isofluorene and inoculated subcutaneously in the right flank with 1 x 106 U87-MG tumor cells suspended in 50 μL PBS. The mice were given potassium iodide at a concentration of 0.1% in their drinking water to block possible free iodide in the drug formulation, with the addition of 0.4% (w/v) sweetener (Sweet and Low) to aid palatability, three days prior to injection and continuing through two weeks post injection. Doses of 95.7µCi and 109.0 µCi of CLR 131were given at Day 0 and Day 7 (N = 8 per group). Control doses of I-127-CLR1404 were given at the indicated dose and was injected via tail vein on Day 0 and Day 7. Results: The control group’s tumors increased 25-fold over a period of four weeks and the treatment group’s tumors increased 12-fold over the same period. There was a significant difference between the control and treatment tumor volumes. Mean survival time was 24.5 days and 31.9 days post injection, respectively. Conclusions: CLR 131 represents a unique, first in class targeted radiotherapeutic currently in clinical development. This study shows that CLR 131 dosed twice provides significant inhibition of tumor growth in a U87-MG tumor model which is known for very aggressive growth. Additionally, CLR 131 provided an overall survival benefit over the control group.

Genetic loci of Mycoplasma agalactiae involved in systemic spreading during experimental intramammary infection of sheep

Mycoplasmas are amongst the most successful pathogens of both humans and animals yet the molecular basis of mycoplasma pathogenesis is poorly understood. This is partly due to the lack of classical virulence factors and little similarity to common bacterial pathogenic determinants. Using Mycoplasma agalactiae as a model we initiated research in this direction by screening a transposon mutant library in the natural sheep host using a negative selection method. Having successfully identified putative factors involved in the colonization of local infection and lymphogenic sites, the current study assessed mutants unable to spread systemically in sheep after experimental intramammary infection. Analysis of distant body sites for complete absence of mutants via SSM PCR revealed that additional set of genes, such as pdhB, oppC, oppB, gtsB, MAG1890, MAG5520 and MAG3650 are required for systemic spreading apart from those that were necessary for initial colonization. Additional in vitro studies with the mutants absent at these systemic sites confirmed the potential role of some of the respective gene products concerning their interaction with host cells. Mutants of pdhB, oppC and MAG4460 exhibited significantly slower growth in the presence of HeLa cells in MEM medium. This first attempt to identify genes exclusively required for systemic spreading provides a basis for further in-depth research to understand the exact mechanism of chronicity and persistence of M. agalactiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0387-0) contains supplementary material, which is available to authorized users.

Solubility-driven toxicity of CuO nanoparticles to Caco2 cells and Escherichia coli: Effect of sonication energy and test environment

Due to small size and high surface energy nanoparticles (NPs) tend to agglomerate and precipitate. To avoid/diminish that, sonication of NPs stock suspensions prior toxicity testing is often applied. Currently, there is no standardized particle sonication protocol available leading to inconsistent toxicity data, especially if toxicity is driven by NPs' dissolution that may be enhanced by sonication.In this study we addressed the effect of sonication on hydrodynamic size (Dh), dissolution and toxicity of copper oxide (CuO) NPs to mammalian cell line Caco-2 in vitro and bacteria Escherichia coli in the respective test environments (cell culture MEM medium, bacterial LB medium and deionised (DI) water). NPs were suspended using no sonication, water bath and probe sonication with different energy intensities.Increased sonication energy (i) decreased the Dh of CuO NPs in all three test environments; (ii) increased dissolution of NPs in MEM medium and their toxicity to Caco-2; (iii) increased dissolution of NPs in LB medium and their bioavailability to E. coli; and (iv) had no effect on dissolution and antibacterial effects of NPs in DI water. Thus, to reduce variations in dissolution and toxicity, we recommend sonication of NPs in DI water following the dilution into suitable test media.

Abstract 3568: Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells

Abstract Targeting the mutant BRAF protein is an accepted approach to the treatment of metastatic melanoma. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. Previous studies have shown that certain isoforms of vitamin E and statins can have synergistic anti-cancer activity. We determined whether a combination of delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor, can exert an anti-neoplastic activity on BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cell lines and whether a differential effect would be evident. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 24 hours then treated with DT3 (0-40 μM), simvastatin (0-5 μM), or a combination and dosed again at 48 hours. SK-MEL-28 and SK-MEL-2 cells grown in 60 mm plates and were treated with DT3 at concentrations of 40, 30, 20 μM, simvastatin at a concentrations of 20, 10, 5 μM or dissolution vehicle as a control for 6 h. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pBAD (Cell Signaling, Danvers, MA). Using MTS assay, we found that DT3 (IC50 38.8 μM) and simvastatin (IC50 22.7μM) have cytotoxic effects on melanoma cell line SK-MEL-28, but on the SK-MEL-2 cells DT3 does not have an effect at the concentrations studied (10-40 μM DT3) yet simvastatin (IC50 16.9 μM) does have cytotoxicity. Further studies determined that combinations of these drugs display a synergistic effect on SK-MEL-28 by inhibition of pS6 and pBAD and subsequent apoptosis. However, these effects are not observed in SK-MEL-2 cells; treated SK-MEL-2 cells show over-expression of Hsp70 and Hsp90 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in BRAF-mutated cells and not in wild type BRAF melanoma cell lines by both DT3 and simvastatin warrants further research into the potential therapeutic use of these combinations. This observation has added importance in the light of recent findings that show the acquisition of BRAF mutation is an early event in melanogenesis and hence these compounds may have a key role in chemoprevention approaches to melanoma. Citation Format: Kelley Cross, Victoria Palau, Marianne Brannon, Janet Lightner, Megan Dycus, William Stone, Koyamangalath Krishnan. Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3568.

Abstract 1346: Magnetoelectric particles cross blood brain barrier to deliver anti-tumor peptide to glioblastoma cells with on-demand release

Abstract Background: Despite significant progress in targeted drug delivery, glioblastoma is one of the least treatable tumors today. Delivering therapy across the blood-brain barrier (BBB) and overcoming resistance of tumor cells to current treatments remains a formidable task. The objective of the current in vitro and in vivo study was to show how a new class of multifunctional nanoparticles termed magnetoelectric nanoparticles (MENs) are used to achieve high-efficacy delivery of a therapeutic load across BBB, then release the load on demand, without affecting normal cells, via application of a specially tailored command sequence of D.C. and A.C. magnetic fields. Methods: Growth hormone (GH)-releasing hormone (GHRH) antagonists, a class of anti-tumor peptides, block the release of tumorigenic insulin-like growth factors. Glioblastoma cell line U87 MG were treated with 1 μM of GHRH antagonist MIA-690 bound to GMO-MENs in MEM media supplemented with 5% FBS and 1% penstrep. In parallel, an in vitro BBB experiment, designed using transwells, applied a D.C. magnetic field gradient to determine optimal field and exposure time for MENs to penetrate the BBB. Next, experimental cells were placed on a D.C. magnetic field (100 Oe) for 12 h to induce nanoelectroporation, then an A.C. field (∼50 Hz) for 2 h to release the peptide intracellularly. Control cells were treated with MIA-690 peptide only, GMO-MENs only, and MIA-690 bound to GMO-MENs at varying magnetic fields and exposure times. Cells were incubated at 37°C with 5% CO2 in a humidified atmosphere. At 24 h and 48 h post-treatment, trypan-blue vital stain assessed cell growth inhibition. Cells were then washed and lysed to measure the concentration of MENs, and MIA-690 using ICP-MS and spectrophotometry, respectively. AFM and SEM-EDS imaged MENs in cell lysate and brain tissue from mice IV injected with MENs, to find signature peaks of Ba and Ti representing the outer core of MENs, to ascertain if particles cross the BBB in vivo. Summary of data: Glioblastoma cells treated with MIA-690 bound to GMO-MENs on a D.C. magnetic field for 12 h, then an A.C. field for 2 h exhibited the greatest penetration of MENs and inhibition of tumor growth. AFM and SEM-EDS imaging showed MENs in cell lysate treated with D.C. for 12 h, but not 2 h or with no magnetic field, confirming application of a low D.C. field for 12 h triggers MENs’ penetration. On-demand MIA-690 intracellular release was highest after exposure to 12 h of D.C. field, then A.C. field at 50 Hz. Confocal microscopy confirmed a transfer of at least 50% of drug-loaded MENs across in vitro BBB, requiring 200 Oe/cm for 5 h. SEM-EDS images of brain slices taken from mice treated with MENs confirmed the presence of Ba and Ti, indicating MENs are able to cross BBB in vivo. Conclusion: Functionalized MENs are effective in treating glioblastomas using targeted, on-demand release of anti-tumor drug with the ability to cross the BBB. Citation Format: Tiffanie Stewart, Emmanuel Stimphil, Rakesh Guduru, Alexandra Rodzinski, Ping Liang, Carolyn Runowicz, Ren-Zhi Cai, Luis Salgueiro, Andrew Schally, Sakhrat Khizroev. Magnetoelectric particles cross blood brain barrier to deliver anti-tumor peptide to glioblastoma cells with on-demand release. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1346.