Successful Rearing of Nutritionally Supplemented Rotifers (Brachionus plicatilis) at Reduced Salinity for Zebrafish (Danio rerio) Polyculture.

Rotifers, Brachionus plicatilis, are a valuable first exogenous feed for zebrafish because they can provide continuous nutrition for growing zebrafish larvae when used in a rotifer-zebrafish polyculture. Typically cultured at high salinities (>10 ppt), B. plicatilis are temporarily immobilized when moved to lower salinities (5 ppt) used for polycultures, decreasing their accessibility and attractiveness to the larvae. The nutritional value of rotifers varies based on their diet, typically live algae, which has limited nutritional value and may pose biosecurity risks. After confirming that rotifers consume and can reproduce when fed an irradiated, processed larval fish diet (PD), they were reared at 5 or 15 ppt, and fed various combinations of an algae mix and/or PD. Population densities and percentages of egg-bearing rotifers were quantified daily until the population density plateaued, and then their nutritional value was assessed. Results indicated that rotifers thrived at both salinities. Those fed PD were successfully maintained at >500 rotifers per mL and contained a greater ω-6/ω-3 fatty acid ratio. Our findings indicate that enriching rotifers with PD raised at 5 ppt can potentially eliminate rotifer immobilization in polyculture, while providing a nutritious, attractive diet for zebrafish larvae and decreasing biosecurity risks.

Abilities of ß-Estradiol to interact with chemotherapeutic drugs, signal transduction inhibitors and nutraceuticals and alter the proliferation of pancreatic cancer cells.

Improving the effects of chemotherapy and reducing the side effects are important goals in cancer research. Various approaches have been examined to enhance the effectiveness of chemotherapy. For example, signal transduction inhibitors or hormonal based approaches have been included with chemo- or radio-therapy. MIA-PaCa-2 and BxPC-3 pancreatic ductal adenocarcinoma (PDAC) cells both express the estrogen receptor (ER). The effects of ß-estradiol on the growth of PDAC cells has not been examined yet the ER is expressed in PDAC cells. We have examined the effects of combining ß-estradiol with chemotherapeutic drugs, signal transcription inhibitors, natural products and nutraceuticals on PDAC. In most cases, inclusion of ß-estradiol with chemotherapeutic drugs increased chemosensitivity. These results indicate some approaches involving ß-estradiol which may be used to increase the effectiveness of chemotherapeutic and other drugs on the growth of PDAC.

Abilities of berberine and chemically modified berberines to interact with metformin and inhibit proliferation of pancreatic cancer cells.

Pancreatic cancer is devastating cancer worldwide with few if any truly effective therapies. Pancreatic cancer has an increasing incidence and may become the second leading cause of death from cancer. Novel, more effective therapeutic approaches are needed as pancreatic cancer patients usually survive for less than a year after being diagnosed. Control of blood sugar levels by the prescription drug metformin in diseases such as diabetes mellitus has been examined in association with pancreatic cancer. While the clinical trials remain inconclusive, there is hope that certain diets and medications may affect positively the outcomes of patients with pancreatic and other cancers. Other natural compounds may share some of the effects of metformin. One "medicinal" fruit consumed by millions worldwide is berberine (BBR). Metformin and BBR both activate AMP-activated protein kinase (AMPK) which is a key mediator of glucose metabolism. Glucose metabolism has been shown to be very important in cancer and its significance is increasing. In the following studies, we have examined the effects of metformin, BBR and a panel of modified BBRs (NAX compounds) and chemotherapeutic drugs on the growth of four different human pancreatic adenocarcinoma cell lines (PDAC). Interestingly, the effects of metformin could be enhanced by BBR and certain modified BBRs. Upon restoration of WT-TP53 activity in MIA-PaCa-2 cells, an altered sensitivity to the combination of certain NAX compounds and metformin was observed compared to the parental cells which normally lack WT-TP53. Certain NAX compounds may interact with WT-TP53 and metformin treatment to alter the expression of key molecules involved in cell growth. These results suggest a therapeutic approach by combining certain pharmaceutical drugs and nutraceuticals to suppress the growth of cancer cells.

Effects of the MDM-2 inhibitor Nutlin-3a on PDAC cells containing and lacking WT-TP53 on sensitivity to chemotherapy, signal transduction inhibitors and nutraceuticals.

Mutations at the TP53 gene are readily detected (approximately 50-75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT-TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT-TP53 activity can have in PDAC cells which normally lack WT-TP53, as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents.

Introduction of WT-TP53 into pancreatic cancer cells alters sensitivity to chemotherapeutic drugs, targeted therapeutics and nutraceuticals.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, highly metastatic malignancy and accounts for 85% of pancreatic cancers. PDAC patients have poor prognosis with a five-year survival of only 5-10%. Mutations at the TP53 gene are readily detected in pancreatic tumors isolated from PDAC patients. We have investigated the effects of restoration of wild-type (WT) TP53 activity on the sensitivity of pancreatic cancer cells to: chemotherapy, targeted therapy, as well as, nutraceuticals. Upon introduction of the WT-TP53 gene into the MIA-PaCa-2 pancreatic cancer cell line, the sensitivity to drugs used to treat pancreatic cancer cells such as: gemcitabine, fluorouracil (5FU), cisplatin, irinotecan, oxaliplatin, and paclitaxel increased significantly. Likewise, the sensitivity to drugs used to treat other cancers such as: doxorubicin, mitoxantrone, and 4 hydroxy tamoxifen (4HT) also increased upon introduction of WT-TP53 into MIA-PaCa-2 cells. Furthermore, the sensitivity to certain inhibitors which target: PI3K/mTORC1, PDK1, SRC, GSK-3, and biochemical processes such as proteasomal degradation and the nutraceutical berberine as increased upon introduction of WT-TP53. Furthermore, in some cases, cells with WT-TP53 were more sensitive to the combination of drugs and suboptimal doses of the MDM2 inhibitor nutlin-3a. However, TP53-independent effects of nutlin-3a were observed upon treatment with either a proteasomal or a PI3K/mTOR inhibitor. These studies indicate the sensitizing effects that WT-TP53 can have in PDAC cells which normally lack WT-TP53 to various therapeutic agents and suggest approaches to improve PDAC therapy.

Metformin influences drug sensitivity in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, highly metastatic malignancy and accounts for 85% of pancreatic cancers. PDAC patients have poor prognosis with a five-year survival of only 5-10% after diagnosis and treatment. Pancreatic cancer has been associated with type II diabetes as the frequency of recently diagnosed diabetics that develop pancreatic cancer within a 10-year period of initial diagnosis of diabetes in increased in comparison to non-diabetic patients. Metformin is a very frequently prescribed drug used to treat type II diabetes. Metformin acts in part by stimulating AMP-kinase (AMPK) and results in the suppression of mTORC1 activity and the induction of autophagy. In the following studies, we have examined the effects of metformin in the presence of various chemotherapeutic drugs, signal transduction inhibitors and natural products on the growth of three different PDAC lines. Metformin, by itself, was not effective at suppressing growth of the pancreatic cancer cell lines at concentration less than 1000 nM, however, in certain PDAC lines, a suboptimal dose of metformin (250 nM) potentiated the effects of various chemotherapeutic drugs used to treat pancreatic cancer (e.g., gemcitabine, cisplatin, 5-fluorouracil) and other cancer types (e.g., doxorubicin, docetaxel). Furthermore, metformin could increase anti-proliferative effects of mTORC1 and PI3K/mTOR inhibitors as well as natural products such as berberine and the anti-malarial drug chloroquine in certain PDAC lines. Thus, metformin can enhance the effects of certain drugs and signal transduction inhibitors which are used to treat pancreatic and various other cancers.

Effects of berberine, curcumin, resveratrol alone and in combination with chemotherapeutic drugs and signal transduction inhibitors on cancer cells-Power of nutraceuticals.

Over the past fifty years, society has become aware of the importance of a healthy diet in terms of human fitness and longevity. More recently, the concept of the beneficial effects of certain components of our diet and other compounds, that are consumed often by different cultures in various parts of the world, has become apparent. These "healthy" components of our diet are often referred to as nutraceuticals and they can prevent/suppress: aging, bacterial, fungal and viral infections, diabetes, inflammation, metabolic disorders and cardiovascular diseases and have other health-enhancing effects. Moreover, they are now often being investigated because of their anti-cancer properties/potentials. Understanding the effects of various natural products on cancer cells may enhance their usage as anti-proliferative agents which may be beneficial for many health problems. In this manuscript, we discuss and demonstrate how certain nutraceuticals may enhance other anti-cancer drugs to suppress proliferation of cancer cells.

Regulation of GSK-3 activity by curcumin, berberine and resveratrol: Potential effects on multiple diseases.

Natural products or nutraceuticals promote anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway. This review will focus on the effects of curcumin (CUR), berberine (BBR) and resveratrol (RES), on the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway, with a special focus on GSK-3. These natural products may regulate the pathway by multiple mechanisms including: reactive oxygen species (ROS), cytokine receptors, mirco-RNAs (miRs) and many others. CUR is present the root of turmeric (Curcuma longa). CUR is used in the treatment of many disorders, especially in those involving inflammatory processes which may contribute to abnormal proliferation and promote cancer growth. BBR is also isolated from various plants (Berberis coptis and others) and is used in traditional medicine to treat multiple diseases/conditions including: diabetes, hyperlipidemia, cancer and bacterial infections. RES is present in red grapes, other fruits and berries such as blueberries and raspberries. RES may have some anti-diabetic and anti-cancer effects. Understanding the effects of these natural products on the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway may enhance their usage as anti-proliferative agent which may be beneficial for many health problems.

Effects of resveratrol, curcumin, berberine and other nutraceuticals on aging, cancer development, cancer stem cells and microRNAs.

Natural products or nutraceuticals have been shown to elicit anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of microRNA (miR) expression which results in cell death or prevents aging, diabetes, cardiovascular and other diseases. This review will focus on a few natural products, especially on resveratrol (RES), curcumin (CUR) and berberine (BBR). RES is obtained from the skins of grapes and other fruits and berries. RES may extend human lifespan by activating the sirtuins and SIRT1 molecules. CUR is isolated from the root of turmeric (Curcuma longa). CUR is currently used in the treatment of many disorders, especially in those involving an inflammatory process. CUR and modified derivatives have been shown to have potent anti-cancer effects, especially on cancer stem cells (CSC). BBR is also isolated from various plants (e.g., Coptis chinensis) and has been used for centuries in traditional medicine to treat diseases such as adult- onset diabetes. Understanding the benefits of these and other nutraceuticals may result in approaches to improve human health.

Cytotoxic and pathogenic properties of Klebsiella oxytoca isolated from laboratory animals.

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.