Nutrient Mixture Research Articles

Long-Term Propagation of Porcine Undifferentiated Spermatogonia.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.

Modulation of MMP-2 and −9 secretion by cytokines, inducers and inhibitors in human melanoma A-2058 cells

Melanoma, an extremely aggressive cancer, causes the most skin cancer-related deaths, due to metastasis to other areas of the body, such as lymph nodes, lungs, liver, brain or bone. It is characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the extracellular matrix and basement membrane, allowing cancer cells to spread to distal organs. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in regulation of MMP-2 and -9 in human melanoma A-2058 cells. Human A-2058 cells were grown in DMEM supplemented with 15% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS and incubated in serum-free media with phorbol 12-myristate 13-acetate (PMA) at 10, 25, 50 and 100ng/ml; TNF-α and IL-1β at 0.1, 1, 10 and 25ng/ml; LPS at 10, 25, 50 and 100µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000µg/ml; actinomycinD and cyclohexamide at 2 and 4µM; retinoic acid and dexamethasone at 50µM. After 24h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Melanoma A-2058 demonstrated strong expression of MMP-2 and slight expression of MMP-9. PMA at 100ng/ml showed no effect on MMP-2 secretion but potently upregulated MMP-9 secretion to 400% that of control. TNF-α showed no significant overall effect on expression of MMP-2 but potent dose-dependent increased MMP-9 secretion with 200% of control at 25ng/ml. IL-1β showed no significant effect on MMP-2 or MMP-9 secretion by A-2058 cells, except at 25ng/ml where MMP-2 level was reduced by ~40% and MMP-9 secretion ~50%. LPS treatment showed no significant effect on MMP-2 secretion and enhanced MMP-9 secretion up to 25µg/ml followed by decreased level. EGCG, NM and doxycycline, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. Actinomycin D, cyclohexamide and retinoic acid had inhibitory effects on MMP-2, while dexamethasone showed slight stimulatory effect on MMP-2 secretion. Our results showed that select cytokines, mitogens and inhibitors modulated A-2058 MMP-2 and MMP-9 expression. They suggest the clinical potential of MMP inhibitors, especially the non-toxic ones, such as the nutrient mixture and its component EGCG in management of melanoma.

319 - A Double-Blind Randomized Cross-Over Study to Investigate the Effect of an Encapsulated Nutrient Mixture on Food Intake and Satiety

319 - A Double-Blind Randomized Cross-Over Study to Investigate the Effect of an Encapsulated Nutrient Mixture on Food Intake and Satiety

A Specific Mixture of Nutrients Suppresses Ovarian Cancer A-2780 Tumor Incidence, Growth, and Metastasis to Lungs.

Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. Despite advances made in chemotherapy and surgery, the average time of clinical remission is approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a specific nutrient mixture (EPQ) containing ascorbic acid, lysine, proline, green tea extract, and quercetin on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n = 12) were all inoculated intraperitoneally (IP) with 2 × 106 cells in 0.1 mL of phosphate buffered saline (PBS) and randomly divided into two groups. Upon injection, the Control group (n = 6) was fed a regular diet and the EPQ group (n = 6) a regular diet supplemented with 0.5% EPQ. Four weeks later, the mice were sacrificed and tumors that developed in the ovary were excised, weighed, and processed for histology. Lungs were inspected for metastasis. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with EPQ in triplicate at concentrations between 0 and 1000 μg/mL. Cell proliferation was measured via MTT assay, MMP-9 secretion via gelatinase zymography, invasion through Matrigel and morphology via hematoxylin and eosin (H & E) staining. All Control mice developed large ovarian tumors, whereas 5 out of 6 mice in the EPQ group developed no tumors, and one, a small tumor. Control mice also showed lung metastasis in 6 out of 6 mice, while no lung metastasis was evident in EPQ mice. Zymography demonstrated only MMP-9 expression, which EPQ inhibited in a dose-dependent fashion, with virtual total block at 250 μg/mL concentration. EPQ significantly inhibited invasion through Matrigel with total block at 250 μg/mL concentration. MTT showed dose-dependent inhibition of cell proliferation with EPQ, and H & E staining showed no morphological changes below 500 μg/mL EPQ. These results suggest that EPQ has therapeutic potential in the treatment of ovarian cancer by significantly suppressing ovarian tumor incidence and growth and lung metastasis, and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells.

Effects of glucose on the uptake and metabolism of glycine in pakchoi (Brassica chinensis L.) exposed to various nitrogen sources

BackgroundPlants can absorb amino acids as a nitrogen (N) source, and glucose is an important part of root rhizodeposition and the soil sugar pool, which participates in the regulation of plant growth and uptake. In pakchoi, the effect of glucose concentration on the glycine N uptake from a nutrient mixture composed of glycine, ammonium, and nitrate, or from a single N solution of glycine alone was studied using specific substrate 15N-labeling and 15N-gas chromatography mass spectrometry.ResultsThe optimal glucose concentration for plant growth was 4.5 μM or 25 μM when supplied with glycine alone or the N mixture, respectively, and resulted in a >25% increase in seedling biomass. The addition of glucose affected the relative contribution from organic or inorganic sources to overall N uptake. When glucose was added at optimal concentrations, glycine was preferentially used as an N source, while the relative contribution from nitrate was reduced. The limiting step for glycine N contribution was active uptake in the roots in high glucose and single-N-source conditions; however, root metabolism of glycine to serine was limiting in high-glucose and mixed-N-source conditions.ConclusionsThe addition of low concentrations of glucose increased the relative uptake of organic nitrogen and reduced the uptake of nitrate, suggesting a feasible way to decrease nitrate content and increase the edible quality of vegetables.

Induction of different activated phenotypes of mouse peritoneal macrophages grown in different tissue culture media.

The role of activated macrophages in the host defense against pathogens or tumor cells has been investigated extensively. Many researchers have been using various culture media in in vitro experiments using macrophages. We previously reported that J774.1/JA-4 macrophage-like cells showed great differences in their activated macrophage phenotypes, such as production of reactive oxygen, nitric oxide (NO) or cytokines depending on the culture medium used, either F-12 (Ham's F-12 nutrient mixture) or Dulbecco modified Eagle's medium (DMEM). To examine whether a difference in the culture medium would influence the functions of primary macrophages, we used BALB/c mouse peritoneal macrophages in this study. Among the activated macrophage phenotypes, the expression of inducible NO synthase in LPS- and/or IFN-γ-treated peritoneal macrophages showed the most remarkable differences between F-12 and DMEM; i.e., NO production by LPS- and/or IFN-γ-treated cells was far lower in DMEM than in F-12. Similar results were obtained with C57BL mouse peritoneal macrophages. Besides, dilution of F-12 medium with saline resulted in a slight decrease in NO production, whereas that of DMEM with saline resulted in a significant increase, suggesting the possibility that DMEM contained some inhibitory factor(s) for NO production. However, such a difference in NO production was not observed when macrophage-like cell lines were examined. These results suggest that phenotypes of primary macrophages could be changed significantly with respect to host inflammatory responses by the surrounding environment including nutritional factors and that these altered macrophage phenotypes might influence the biological host defense.

Short communication: Arginase inhibition reduces the synthesis of casein in bovine mammary epithelial cells

The main purpose of this work was to determine the effect of arginase inhibition via Nω-hydroxy-nor-l-arginine (nor-NOHA) on casein synthesis in bovine mammary epithelial cells (BMEC). Passage 2 BMEC isolated from dairy cows were seeded to 6-well plates and randomly divided into 4 treatments: (1) control [Dulbecco's modified Eagle medium:Nutrient Mixture F-12 medium (DMEM/F12)]; (2) nor-NOHA (DMEM/F12 + 1 mmol/L nor-NOHA); (3) nor-NOHA + arginine (DMEM/F12 + 1 mmol/L nor-NOHA + 3.2 mmol/L Arg); and (4) nor-NOHA + ornithine (DMEM/F12+ 1 mmol/L nor-NOHA + 1 mmol/L Orn). Then, we determined the activity of enzymes related to Arg metabolism and casein synthesis in BMEC and the proliferation of cells. The addition of nor-NOHA reduced the activity of arginase and ornithine decarboxylase but had no effect on the activity of nitric oxide synthase, and these responses were the same at the gene expression level. In addition, supplementation of nor-NOHA in BMEC reduced cellular proliferation and casein synthesis. Addition of Arg to nor-NOHA resulted in cellular proliferation and casein synthesis similar to that of nor-NOHA alone. In contrast, addition of Orn to the medium with nor-NOHA increased the synthesis of casein and cellular proliferation compared with Nor-NOHA. In conclusion, suppression of the Arg-arginase-Orn pathway reduced casein synthesis and cellular proliferation, which indicated that this pathway is an important regulator of the synthesis of casein in BMEC.

Abstract P3-05-07: Flutamide reduced tumor progression and altered steroid hormone secretion in human and canine inflammatory breast cancer cell lines

Abstract Background: Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate and accounts for 6% of diagnosed breast cancers. The role of androgens on breast cancer is on rise in research, trying to propose anti-androgen therapeutic strategies. The aim of this study was to determine the effects in vivo and in vitro of flutamide (anti-androgen drug) on cell proliferation, tumor progression and steroid production in two cancer IBC triple negative cell lines (SUM-149 and IPC-366, human and canine, respectively). Material and Methods: IPC-366 was cultured in Dulbecco's modified Eagle medium nutrient mixture F-12 Ham (DMEM/F12) and SUM149 was maintained in Ham's F-12 media. Flutamide concentrations added to the culture media were: 5 µm, 10 µm, and 15 µm for 72 hours. Additionally, IPC-366 and SUM149 xenotrasplanted mice were used for in vivo assays with the same flutamide concentrations administrated subcutaneously. Steroid hormones determination in culture media and tumor homogenates (pregnenolone (P5), progesterone (P4), dihydroepiandrostenedione (DHEA), androstenedione (A4), testosterone (T), dihydritestosterone (DHT), 17β-estradiol (E2) and estrone sulphate (SO4E1)) were assayed by EIA previously validated. Immunohistochemical (IHC) analysis of the steroidogenic enzymes CYP11A1, 3β-HSD, CYP19A1, 17β-HSD and 5α-reductase were assayed. Results: Percentage of cell proliferation showed a decrease in all treatments in IPC-366 and SUM149. In vivo tumor progression was reduced in around 65% in IPC-366 and SUM149 xenotrasplanted mice. Regarding hormonal secretion assayed in pellets and homogenates. in treated groups there was an increased in steroid secretion as showed the high levels found in P5, P4 and A4. T and DHT concentrations were higher in treated groups, in contrast to E2 levels that decreased. 17β-HSD and 5α-reductase by IHC showed a high expression in treated groups. Conclusion: IPC-366 and SUM149 treated with flutamide reduced the proliferation of neoplastic cells, reduced tumor progression in xenotrasplanted mice and altered steroid hormone secretion by increasing T production and decreasing in E2 levels. These results open a future approach for IBC and triple negative breast cancer. Citation Format: Caceres S, Pena L, Silvan G, Illera MJ, Monsalve B, Woodward WA, Reuben J, Illera JC. Flutamide reduced tumor progression and altered steroid hormone secretion in human and canine inflammatory breast cancer cell lines [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-07.

Modulation of MMP-2 and MMP-9 secretion by cytokines, inducers and inhibitors in human glioblastoma T-98G cells.

Brain tumors are highly aggressive, characterized by the secretion of high levels of matrix metalloproteinase (MMP)-2 and MMP-9 that degrade the extracellular matrix and basement membrane, allowing cancer cells to spread to distal organs. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activity. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human glioblastoma T-98G cells. Human T-98G cells were grown in DME supplemented with 15% fetal bovine serum and antibiotics in 24-well tissue culture plates. At near confluence, cells were washed with phosphate-buffered saline and incubated in serum-free media with: phorbol12-myristate 13-acetate (PMA) at 10, 25, 50 and 100ng/ml; tumor necrosis factor (TNF)-α and interleukin (IL)-1β at 0.1, 1, 10 and 25ng/ml; lipopolysaccharide (LPS) at 10, 25, 50 and 100µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000µg/ml; actinomycinD and cyclohexamide at 2and4µM; retinoic acid and dexamethasone at 50µM. After 24h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Glioblastoma T-98G cells expressed only one band corresponding to MMP-2. PMA treatment showed increased MMP-2 and MMP-9 secretions up to 25ng/ml and decreased levels of secretions at 50and100ng/ml, with no significant overall effect. TNF-α induced an up and down effect on MMP-2 and a slight induction of MMP-9. IL-1β demonstrated a slight dose-dependent increase in T-98G secretion of MMP-2, but no induction of MMP-9. LPS showed dose-dependent decreased inactive MMP-2 secretion, increased active MMP-2 secretion and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. ActinomycinD, cyclohexamide, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated T-98G cell MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the nutrient mixture and its component EGCG in the management of glioblastoma cancers.

Cytokines, inducers and inhibitors modulate MMP-2 and MMP‑9 secretion by human Fanconi anemia immortalized fibroblasts.

Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated in tumor invasion and metastasis. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human immortalized fibroblasts from FA. Human FA immortalized fibroblast cell lines FA-A:PD220 and FA-D2:PD20 were grown in minimum essential medium (MEM) supplemented with 15% fetal bovine serum (FBS) and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with phosphate‑buffered saline (PBS) and incubated in serum-free media with the following: phorbol 12-myristate 13-acetate (PMA) at 10-100ng/ml; tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) at 0.1-25ng/ml; lipopolysaccharide (LPS) at 10-100µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10-100µM without and with PMA; a nutrient mixture (NM) without and with PMA at 10-1,000µg/ml; actinomycin-D and cyclohexamide at 2and4µM; retinoic acid and dexamethasone at 50µM. After 24h, media were removed and analyzed for MMP-2 and MMP-9 by zymography. Both FA cell lines expressed only MMP-2 and responded similarly to cytokines, mitogens, inducers and inhibitors. PMA potently stimulated MMP-9 and had a moderate effect on MMP-2. TNF-α showed variable effects on MMP-2 and significantly enhanced MMP-9. IL-1β enhanced MMP-2 slightly and MMP-9 significantly. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated MMP-2 and MMP-9 expression. Actinomycin-D, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the NM and its component EGCG in the management of FA cancers.

A Novel Organ Culture Model to Quantify Collagen Remodeling in Tree Shrew Sclera.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 μL and (iv) 200 μL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.

Sampling for MEOR: Comparison of surface and subsurface sampling and its impact on field applications

The increasing demand for oil and also increasing numbers of mature oil reservoirs have attracted attention to enhanced oil recovery (EOR) in which microbial enhanced oil recovery (MEOR) is one of the commercial options. In order to develop a MEOR strategy for a given field it is extremely important to evaluate the microbial community present, as this will be the basis for a customized nutrient mixture. The objective of this study was to assess how sampling strategies (surface vs. subsurface) can accurately reflect the microbial community and microbial activity in a reservoir. In addition to fluid samples the analysis of sand deposits, also collected from the well bottom, was included to get insight into presence and identity of attached microbes. Fluid samples were collected at the wellhead (surface) and in-situ at a depth of approximately 780m tvdss (true vertical depth subsea) under a reservoir pressure of 31 bars (subsurface). The pressure was released either quickly (within minutes) or slowly (stepwise, within 11 days) to assess the potential activity loss of microorganisms sensitive towards rapid pressure changes. Quantitative PCR (polymerase chain reaction) for determining cell numbers showed higher amounts of microbial cells in the subsurface sample compared to the surface water. The composition of the microbial community was only slightly more diverse in the subsurface sample than in the surface sample. Sand material, however, showed the most diverse community. Biogenic CO2 formation rates were determined under atmospheric and high pressure conditions with highest rates, i.e. highest microbial activity, in the wellhead fluid and the solid sand sample. In conclusion no substantial differences were observed between fast and slowly depressurized subsurface fluids and also between surface and subsurface fluid samples in terms of microbiological activity and diversity. Therefore, using wellhead fluids for microbiological analysis and MEOR development seems to be a valid option although it has always to be carefully considered not to miss out on information of solid samples regarding various aspects such as community and biofilm formation.

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

Abstract 4053: A unique nutrient mixture suppresses ovarian cancer growth of A-2780 by inhibiting invasion and MMP-9 secretion

Abstract Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. The American Cancer Society estimated that it would claim 14,250 lives in 2013. Despite the advances made in chemotherapy and surgery, the average time of clinical remission is approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a unique nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n = 12) were inoculated by I.P. with 2×106 cells in 0.1ml PBS and randomly divided into two groups. Group A (n = 6) was fed a regular diet and group B (n = 6) a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and tumors that developed in the ovary were excised, weighed and processed for histology. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with NM in triplicate at concentrations between 0-1000 μg/ml. Cell proliferation was measured by MTT assay, MMP-9 secretion by gelatinase zymography, invasion through Matrigel and morphology by H&E staining. All control mice (Group A) developed large ovarian tumors, whereas 5 out of 6 mice in group B developed no tumors, and one, a small tumor. Zymography demonstrated only MMP-9 expression, which NM inhibited in a dose dependent fashion, with virtual total block at 250 μg/ml concentration. NM significantly inhibited invasion through Matrigel with total block at 250 μg/ml concentration. MTT showed dose dependent inhibition of cell proliferation with NM and H&E staining showed no morphological changes below 500 μg/ml NM. These results suggest that NM has therapeutic potential in treatment of ovarian cancer by significantly suppressing tumor growth and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells. Citation Format: M. Waheed Roomi, Matthias Rath, Aleksandra Niedzwiecki. A unique nutrient mixture suppresses ovarian cancer growth of A-2780 by inhibiting invasion and MMP-9 secretion. [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 4053.

Ginsenosides Rg1 enhance the ability of olfactory ensheathing cells to repair spinal cord injury: An experimental study

Objective To study the effects of ginsenoside Rg1 on effects of olfactory ensheathing cells (OECs) in spinal cord injury as well as their expression of neurotrophic factors like ciliary neurotrophic factor (CNTF), vascular endothelial growth factor (VEGF). Methods OECs were primary cultured and purified from olfactory bulb of newborn SD rats. Purified cells were divided into 2 groups, the experimental group was treated with Rg1 in concentration of 40 μg/ml for 72 h (the optimal concentration and action time which have been proven by our previous study), the control group received an equal volume of Dulbecco's Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) medium. The models of spinal cord injury was established by using Allen's combat, and were randomly divided into 3 groups, OECs group (inject the control group cells), Rg1+ OECs group (inject the experimental group cells) and control group (inject an equal volume of DMEM/F12 medium). Using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine mRNA expression of CNTF and VEGF in OECs group and Rg1+ OECs group. Results The Basso Beattie Bresnahan (BBB) score of Rg1+ OECs group was higher than the other two groups (15.0±1.0 vs.12.0±2.0, 7.4±0.9, P<0.05). Histology revealed that Rg1+ OECs group had larger number of neurons (64±3, 38±2, 26±2, P<0.01), lower level of GFAP expression and less damage of spinal cord tissue than OECs group and control group (8.65±0.32, 4.93±0.25, 2.21±0.17, P<0.01). RT-PCR analysis demonstrated that Rg1 significantly up-regulated mRNA expression of CNTF and VEGF (0.78±0.03, 0.39±0.03; 0.86±0.03, 0.61±0.02, P<0.05). Conclusion Ginsinoside Rg1 obviously improve the effects of olfactory ensheathing cell transplantation for spinal cord injury by the up-regulation of CNTF and VEGF. Key words: Ginsenoside Rg1; Olfactory ensheathing cells; Spinal cord injury; Cell transplantation; Gene expression

Suspension Culture Alters Insulin Secretion in Induced Human Umbilical Cord Matrix-Derived Mesenchymal Cells.

Worldwide, diabetes mellitus (DM) is an ever-increasing metabolic disorder. A promising approach to the treatment of DM is the implantation of insulin producing cells (IPC) that have been derived from various stem cells. Culture conditions play a pivotal role in the quality and quantity of the differentiated cells. In this experimental study, we have applied various culture conditions to differentiate human umbilical cord matrix-derived mesenchymal cells (hUCMs) into IPCs and measured insulin production. In this experimental study, we exposed hUCMs cells to pancreatic medium and differentiated them into IPCs in monolayer and suspension cultures. Pancreatic medium consisted of serum-free Dulbecco's modified eagle's medium Nutrient mixture F12 (DMEM/F12) medium with 17.5 mM glucose supplemented by 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, and B-27 serum-free supplement. After differentiation, insulin content was analyzed by gene expression, immunocytochemistry (IHC) and the chemiluminesence immunoassay (CLIA). Reverse transcription-polymerase chain reaction (RT-PCR) showed efficient expressions of NKX2.2, PDX1 and INSULIN genes in both groups. IHC analysis showed higher expression of insulin protein in the hanging drop group, and CLIA revealed a significant higher insulin production in hanging drops compared with the monolayer group following the glucose challenge test. We showed by this novel, simple technique that the suspension culture played an important role in differentiation of hUCMs into IPC. This culture was more efficient than the conventional culture method commonly used in IPC differentiation and cultivation.

What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell–cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.

A NUTRIENT MIXTURE INHIBITS GLIOBLASTOMA XENOGRAFT U-87 MG GROWTH IN MALE NUDE MICE

Brain tumors are highly aggressive tumors characterized by secretions of high levels of matrix metalloproteinase-2 and -9, leading to tumor growth, invasion and metastasis by digesting the basement membrane and extracellular matrix components. We previously demonstrated the effectiveness of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract in vitro: on activity of urokinase plasminogen activator, matrix metalloproteinases and TIMPs in various human glioblastoma (LN-18, T-98G and A-172) cell lines and on glioblastoma A-172 cell proliferation and Matrigel invasion. Our main objective in this study was to investigate the effect of the NM in vivo on human glioblastoma U-87 MG cell line. Athymic male nude mice inoculated with 3·10(6) U-87 MG cells subcutaneously and were fed a regular diet or a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed, the tumors were weighed and measured. The samples were studied histologically. NM inhibited tumor weight and tumor burden by 53% (p = 0.015) and 48% (p = 0.010), respectively. These results suggest the therapeutic potential of NM as an adjuvant in the treatment of glioblastoma.

Effect of a Nutrient Mixture on Fanconi Anemia Fibroblast and Normal Human Dermal Fibroblast: A Comparison

Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays certain abnormalities as compared to normal human dermal fibroblast (NHDF). This prompted us to investigate the effect of a specific nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract, which has demonstrated a broad spectrum of pharmacological activities, on FAF compared to NHDF. We investigated the in vitro effect of NM on FAF and NHDF cell proliferation by MTT assay, MMPs secretion by zymography, morphology by H&E staining and apoptosis by green caspase assay. FAF (FA-A: PD20, FA-A: PD220) and NHDF were cultured in modified Dulbecco Eagle media. At near confluence, the cells were treated with different concentrations of NM (0, 50, 100, 250, 500 and 1000 μg/ml) in triplicate. The cells were also treated with PMA to induce MMP-9 activity. NM had no effect on FAF cell viability in both cell lines compared to control. In contrast NM exhibited 20% at 50 and 100, 50% at 250, 60% at 500 and 70% toxicity at 1000 μg/ml on NHDF cells. Zymography demonstrated MMP-2 and MMP-9 on PMA stimulation in FAF and NM inhibited the activity of both MMP-2 and MMP-9 in a dose response fashion with total block at 500 μg/ml. In contrast, NHDF exhibited only MMP-2, both active and inactive forms, and NM inhibited their activities in a dose-dependent manner with total block at 1000 μg/ml. H&E staining did not indicate any morphological changes in FAF nor induced apoptosis at higher concentrations, as seen by caspases assay. However, although no morphological changes in NHDF were noted up to NM 100 μg/ml, progressive changes in cell shrinkage, rounding and nuclear condensation, pertaining to apoptosis, were observed at higher concentrations. These changes were consistent with the results from the green caspases apoptosis assay. Our data demonstrate that NM exhibited different responses toward FAF and NHDF. This may in part be due to elevated chromosomal break, deletion and hypersensitivity to cross linking agents, a DNA repair disorder in FAF that is lacking in NHDF.

Nutritional Status and Blood Biochemical Parameters in Anoestrus Crossbred Cattle in Malwa Region of Madhya Pradesh

Reproductive failure is a major problem in crossbred cattle in Malwa region of Madhya Pradesh. To find out the nutritional causes behind anoestrus, thirty anoestrus crossbred cattle including 20 heifers (BW 224.75±7.41 kg) and 10 cows (BW 316.50±15.48 kg; milk yield 4.9±1.25 L/h/d) were randomly selected from ten villages of Indore district. The average daily feed intake of each animal was recorded and proximate principles, major elements (Ca and P) and trace elements (Fe, Zn, Mn, Cu and Co) in the available feedstuffs were determined to find out nutrient availability. Deficiency of various nutrients was calculated by comparing availability with standard requirements of the animals. Blood samples were also collected and analyzed for different haemato-biochemical constituents. The average values for Hb 9.19±0.20 and 10.34±0.28 g/dL; blood glucose, 49.29±0.91 and 55.73±1.42 mg/dL; plasma protein 5.05±0.09 and 6.80±0.26 g/dL were found in heifers and cows, respectively. Major mineral profile in heifers and cows were Ca, 9.65±0.17 and 8.81±0.29 mg/dL; P 5.12±0.14 and 5.12±0.34 mg/dL; Total P 15.50±1.14 and 24.00±2.21 mg/dL, respectively. Trace mineral levels in heifers and cows were Fe 24.35±2.12 and 18.96±1.43 ppm; Zn 1.45±0.07 and 1.62±0.09 ppm; Cu 0.65±0.06 and 0.91±0.12 ppm; Mn 0.74±0.10 and 0.86±0.07 ppm; Co 1.82±0.30 and 2.54±0.44 ppm, respectively. Results indicated that values of Hb, blood glucose, total protein, Ca, P, Zn were marginally lower but Cu was deficient in heifers while in cows the levels of Ca, P and Zn were marginally lower. Fe levels were found to be high in both heifers and cows. It may be concluded that dietary deficiency reflected the hemato-biochemical profile of anoestrus animals and a nutrient mixture has been prepared for minimization of anoestrus in crossbred cattle.