Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation.

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.

Freshwater clam extract reduces liver injury by lowering cholesterol accumulation, improving dysregulated cholesterol synthesis and alleviating inflammation in high-fat, high-cholesterol and cholic acid diet-induced steatohepatitis in mice.

Freshwater clam (Corbicula fluminea) is a traditional liver-protective food in Asia. Recent studies have renewed attention on high cholesterol accumulation and dysregulated cholesterol synthesis in the liver as a critical factor in the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH). In this study, we investigated the protective effects of freshwater clam extract (FCE) and its fat fraction (FCE oil) on high-fat, high-cholesterol and cholic acid (HFHC) diet-induced lean steatohepatitis in mice. Mice were fed a HFHC diet containing FCE or FCE oil for 6 weeks. FCE, but not FCE oil, feeding reduced liver injury as indicated by decreased plasma alanine aminotransferase activity. Liver total cholesterol accumulation was reduced after FCE and FCE oil treatment. Accumulation of squalene and desmosterol, the precursors of cholesterol, in the liver was reduced by FCE but not by FCE oil. The caspase-1 (p10) and interleukin (IL)-1ß (p17) protein expressions in the liver were suppressed by both FCE and FCE oil. Therefore, FCE may act as functional food that can reduce steatohepatitis and liver injury by reducing cholesterol accumulation, improving dysregulated cholesterol synthesis and attenuating inflammation.

Vascular- and hepato-protective effects of passion fruit seed extract containing piceatannol in chronic high-fat diet-fed rats.

The effects of chronic administration of piceatannol-enriched (9.5% w/w) passion fruit seed extract (PFSE) on the cardiovascular damage induced in a high-fat (HF) diet-fed model of Fischer 344 rats were evaluated. Rats were fed the control, HF, or HF diets containing PFSE (0.5% w/w) for 16 weeks, and the effects of the various diets on the tissue weight, serum lipid profile, hepatic fibrosis, hepatic ductular reaction, cardiac function and aortic ring reactivity were examined. HF diet-fed rats developed signs of cardiovascular disease with abnormal serum profiles compared to control diet-fed rats. PFSE supplementation improved the liver hypertrophy and hepatic histology of the HF diet-fed rats. In addition, the triglyceride and cholesterol levels, platelet aggregation, cardiac function, and acetylcholine-mediated relaxation of the aortic ring were improved. These results suggest that the chronic intake of PFSE containing piceatannol prevents HF diet-induced cardiovascular disease in rats.

Changes in serum proteins after endotoxin administration in healthy and choline-treated calves.

BACKGROUND: This study aimed to investigate the possible serum protein changes after endotoxin administration in healthy and choline-treated calves using proteomics. These results are expected to contribute to the understanding of the pathophysiological mechanisms of endotoxemia and the beneficial effect of choline administration in this clinical situation. METHODS: Healthy-calves (n = 20) were divided into 4 groups: Control, Choline treated (C), Lipopolysaccharide administered (LPS), and LPS + C. Control calves received 0.9 % NaCl injection. Calves in C and LPS + C groups received choline chloride (1 mg/kg/iv). Endotoxin (LPS) was injected (2 µg/kg/iv) to the calves in LPS and LPS + C groups. Serum samples were collected before and after the treatments. Differentially expressed proteins (> 1.5 fold-change relative to controls) were identified by LC-MS/MS. RESULTS: After LPS administration, 14 proteins increased, and 13 proteins decreased within 48 h as compared to controls. In the LPS group, there were significant increases in serum levels of ragulator complex protein (189-fold) and galectin-3-binding protein (10-fold), but transcription factor MafF and corticosteroid binding globulin were down regulated (≥ 5 fold). As compared with the LPS group, in LPS + C group, fibrinogen gamma-B-chain and antithrombin were up-regulated, while hemopexin and histone H4 were down-regulated. Choline treatment attenuated actin alpha cardiac muscle-1 overexpression after LPS. CONCLUSIONS: LPS administration produces changes in serum proteins associated with lipid metabolism, immune and inflammatory response, protein binding/transport, cell adhesion, venous thrombosis, cardiac contractility and blood coagulation. The administration of choline is associated with changes in proteins which can be related with its beneficial effect in this clinical situation.

Lactoferrin attenuates fatty acid-induced lipotoxicity via Akt signaling in hepatocarcinoma cells.

Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). The excess influx of fatty acids (FAs) into the liver is recognized as a main cause of simple steatosis formation and progression to NASH. Recently, administration of lactoferrin (LF), a glycoprotein present in milk, was suggested to prevent NAFLD development. However, the effect of LF on the contribution of FA to NAFLD development remains unclear. In this study, the effects of LF on FA mixture (FAm)-induced lipotoxicity using human hepatocarcinoma G2 cells were assessed. FAm significantly decreased cell viability and increased intracellular lipid accumulation, whereas LF significantly recovered cell viability without affecting lipid accumulation. FAm-induced lactic dehydrogenase (LDH) and caspase-3/7 activities were significantly decreased by LF and SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. We also found that LF added to FAm-treated cells induced Akt phosphorylation, which contributed to inhibition of JNK signaling pathway-dependent apoptosis. Akt inhibitor VIII, an allosteric Akt inhibitor, significantly attenuated the effect of LF on LDH activity and abrogated the ones on cell viability and caspase-3/7 activity. In summary, the present study has revealed that LF has a protective effect on FAm-induced lipotoxicity in a HepG2 model of NAFLD and identified the activation of the Akt signaling pathway as a possibly major mechanism.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation. .

Flavonoids and saponins extracted from black bean (Phaseolus vulgaris L.) seed coats modulate lipid metabolism and biliary cholesterol secretion in C57BL/6 mice.

Black bean (Phaseolus vulgaris L.) seed coats are a rich source of natural compounds with potential beneficial effects on human health. Beans exert hypolipidaemic activity; however, this effect has not been attributed to any particular component, and the underlying mechanisms of action and protein targets remain unknown. The aim of the present study was to identify and quantify primary saponins and flavonoids extracted from black bean seed coats, and to study their effects on lipid metabolism in primary rat hepatocytes and C57BL/6 mice. The methanol extract of black bean seed coats, characterised by a HPLC system with a UV-visible detector and an evaporative light-scattering detector and HPLC-time-of-flight/MS, contained quercetin 3-O-glucoside and soyasaponin Af as the primary flavonoid and saponin, respectively. The extract significantly reduced the expression of SREBP1c, FAS and HMGCR, and stimulated the expression of the reverse cholesterol transporters ABCG5/ABCG8 and CYP7A1 in the liver. In addition, there was an increase in the expression of hepatic PPAR-α. Consequently, there was a decrease in hepatic lipid depots and a significant increase in bile acid secretion. Furthermore, the ingestion of this extract modulated the proportion of lipids that was used as a substrate for energy generation. Thus, the results suggest that the extract of black bean seed coats may decrease hepatic lipogenesis and stimulate cholesterol excretion, in part, via bile acid synthesis.

A systematic review of Gymnema sylvestre in obesity and diabetes management.

The prevalence of obesity is associated with many health-related problems. Currently, more than 300 million people are considered to be obese. According to the World Health Organization (WHO), by 2030, 87 and 439 million people will be affected in India and the world, respectively. Today, herbal medicines are gaining interest in the treatment of obesity and diabetes, because of their minimal side effects. Gymnemic acid - an active component isolated from Gymnema sylvestre - has anti-obesity and antidiabetic properties, decreases body weight and also inhibits glucose absorption. Several components extracted from Gymnema prevent the accumulation of triglycerides in muscle and liver, and also decrease fatty acid accumulation in the circulation. In this paper, an attempt has been made to review the effects of various extracts from Gymnema sylvestre in the regulation of carbohydrate and lipid metabolism in both animal and clinical studies.

An in vitro study reveals nutraceutical properties of Ananas comosus (L.) Merr. var. Mauritius fruit residue beneficial to diabetes.

BACKGROUND: Rapid urbanisation and nutritional transition is fuelling the increased global incidence of type 2 diabetes. Pineapple fruit residue was explored for its nutraceutical properties as an alternative or adjunct to currently available treatment regime. Ethyl acetate and methanolic extracts of pineapple fruit residue were evaluated for anti-diabetic activity in cell free and cell based systems. Specifically, we assessed: (1) antioxidant potential, (2) anti-glycation potential, (3) carbohydrate digestive enzyme inhibition, and (4) lipid accumulation and glycerol-3-phosphate dehydrogenase activity in differentiating 3T3-L1 cells. RESULTS: The active components in the ethyl acetate and methanolic extracts were identified as sinapic acid, daucosterol, 2-methylpropanoate, 2,5-dimethyl-4-hydroxy-3(2H)-furanone, methyl 2-methylbutanoate and triterpenoid ergosterol using DART/HRMS and ESI/HRMS. Micronutrient analysis revealed the presence of magnesium, potassium and calcium. Adipogenic potential, anti-glycation property of the ethyl acetate extract, and DNA damage protection capacity of the methanolic extract are promising. CONCLUSION: Results from this study clearly indicate that pineapple fruit residue could be utilised as a nutraceutical against diabetes and related complications.

Gene expression analysis of the liver and skeletal muscle of psyllium-treated mice.

Psyllium, a dietary fibre rich in soluble components, has both cholesterol- and TAG-lowering effects. Many studies have verified these actions using liver samples, whereas little information is available on the effects of psyllium treatment on other organs. The purpose of the present study was to evaluate the possible beneficial effects of psyllium. We investigated the gene expression profiles of both liver and skeletal muscle using DNA microarrays. C57BL/6J mice were fed a low-fat diet (LFD; 7 % fat), a high-fat diet (HFD; 40 % fat) or a HFD with psyllium (40 % fat+5 % psyllium; HFD+Psy) for 10 weeks. Body weights and food intake were measured weekly. After 10 weeks, the mice were killed and tissues were collected. Adipose tissues were weighed, and plasma total cholesterol and TAG blood glucose levels were measured. The expression levels of genes involved in glycolysis, gluconeogenesis, glucose transport and fatty acid metabolism were measured by DNA microarray in the liver and skeletal muscle. In the HFD+Psy group, plasma total cholesterol, TAG and blood glucose levels significantly decreased. There was a significant reduction in the relative weight of the epididymal and retroperitoneal fat tissue depots in mice fed the HFD+Psy. The expression levels of genes involved in fatty acid oxidation and lipid transport were significantly up-regulated in the skeletal muscle of the HFD+Psy group. This result suggests that psyllium stimulates lipid transport and fatty acid oxidation in the muscle. In conclusion, the present study demonstrates that psyllium can promote lipid consumption in the skeletal muscle; and this effect would create a slightly insufficient glucose state in the liver.

Thermal and refining processes, not fermentation, tend to reduce lipotropic capacity of plant-based foods.

Plant-based foods (PBF) are relevant and diversified sources of lipotropes, which are compounds preventing excess hepatic fat deposits. In a first study, we defined the lipotropic capacity (LC, %) of raw PBF as the means of 8 lipotrope densities (LD, mg/100 kcal), each expressed relative to that of a reference food ranking the highest considering its mean 8 LD ranks (LC(raw asparagus)=100%) (A. Fardet, J.-F. Martin and J. M. Chardigny, J. Food Comp. Anal., 2011, DOI: 10.1016/j.jfca.2011.1003.1013). We showed that vegetables appeared as the best source of lipotropes on a 100 kcal-basis compared to legumes, cereals, fruits and nuts. The main objective of this second study was to quantify the effect of processing on LD and LC of raw PBF based on lipotrope contents collected in a USDA (United State Department of Agriculture) database and the literature, i.e. betaine, choline, myo-inositol, methionine, magnesium, niacin, pantothenic acid and folate contents. Choline and betaine densities were not significantly affected by processing while methionine and lipotropic micronutrient densities were significantly decreased, especially for magnesium, pantothenate and folates. Myo-inositol density decreases were insignificant due to lower product number resulting from limited literature data. Lipotropic micronutrient densities were more affected by processing than other densities. Fermentations increased betaine (median change of +32%) and choline (+34%) densities. Canning and boiling vegetables increased choline densities (+26%). Globally, processing significantly reduced LC by ∼20%, fermentations being less drastic (median change of -5%) than refining (-33%) and thermal treatments (-16%). More specifically, canning increased LC of beetroot (536 vs 390%) and common bean (40 vs 36%) as fermentation towards LC grape (14 vs 7% for wine). Results were then mainly discussed based on percentages of lipotrope content changes on a dry-weight basis. Results of this study also showed that the LC is quite a relevant index to estimate effect of processing on lipotropic potential of PBF.

Assembly of hybrid bacteriophage Qbeta virus-like particles.

Bacteriophage Qbeta coat protein forms uniform virus-like particles when expressed recombinantly in a variety of organisms. We have inserted the IgG-binding Z domain at the carboxy terminus of the coat protein and coexpressed this chimeric subunit with native coat protein to create hybrid, IgG-binding virus-like particles. Extracellular osmolytes were found to have an effect on the efficiency of incorporation of fusion proteins into VLPs in Escherichia coli when a carbenicillin, but not a kanamycin, selection marker was used. The addition of sucrose to the growth medium decreased the incorporation efficiency; the osmoprotectant glycine betaine eliminated this effect. The decrease in efficiency was not observed when carbenicillin was omitted from the final expression culture. The addition of sodium chloride instead of sucrose gave rise to particles with a larger number of fusion proteins than the standard conditions. These results illustrate that cellular conditions should be taken into account even in apparently simple systems when natural or engineered protein nanoparticles are made.

The impact of fatty acid oxidation on energy utilization: targets and therapy.

Utilization of fat as a long-term energy storage vehicle is crucial for the maintenance of cellular metabolism and is under intricate and many times redundant control mechanisms. Aberrations in the control of energy metabolism is apparent in diseases such as diabetes and obesity and is evident early on in patients with impaired glucose tolerance. Insulin resistance has been observed at the level of muscle, liver and adipose tissue. Hyperglycemia is the hallmark of diabetes and is characterized by decreased glucose disposal and increased glucose production, driven by enhanced and uncontrolled fatty acid oxidation (FAO). Mechanisms aimed at limiting the availability of substrates or the activity of processes involved in FAO should provide an immediate reduction in undesired glucose production in these individuals. Numerous targets are available which influence directly the metabolism of fat, including limiting availability of substrate to FAO, inhibiting oxidation of the fatty acid per se, and uncoupling the energy obtained during the oxidation of the fatty acid. These include antilipolytic agents which limit the availability of substrate, FAO inhibitors which limit fatty acid transport (carnitine palmitoyl transferase, CoA sequestration), FAO per se (beta oxidation), and agents which uncouple the energy of FAO (uncoupling proteins, beta3 agonists). These other targets which affect fatty acid metabolism indirectly will be discussed in this review with 184 references.

Functional and pharmacological characterization of human Na(+)-carnitine cotransporter hOCTN2.

L-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for beta-oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na(+)-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage- clamp and flux measurements its functional and pharmacological properties as a Na(+)-carnitine cotransporter. L-carnitine transport was electrogenic. The L-carnitine-induced currents were voltage and Na(+) dependent, with half-maximal currents at 0.3 +/- 0.1 mM Na(+) at -60 mV. Furthermore, L-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na(+)-coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant (K(m)) of 4.8 +/- 0.3 microM for L-carnitine and 98.3 +/- 38.0 microM for D-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC(50) for emetine was 4.2 +/- 1.2 microM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, pointing to a different mode of action. In summary, hOCTN2 mediates electrogenic Na(+)-dependent stereoselective high-affinity transport of L-carnitine and Na(+). hOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency.

The positive role of voids in the plasma membrane in growth and energetics of Escherichia coli.

Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration. Permeability of hydrogen peroxide, a non-electrolyte of molecular size between water and urea, through the bacterial membranes changed directly with the rate of respiration (an activity residing in the bacterial plasma membrane) in E. coli and the enhanced permeability and respiratory activity were highly correlated. Hydrogen peroxide permeability and induction of voids (spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these cavities) were shown to be a direct and excellent measure of leak conductance. Fluorescence intensity and anisotropy of the extrinsic fluorescent probes (incorporated by growing bacteria in their presence) decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition. The physical basis of osmotic phenomena, as a relevant (thermodynamic) volume, could relate to water exchange or compression, depending on the osmotic domain. In the domain of compression in bacteria, i.e. well above the isotonic range, the computed activation volume was consistent with voids in the membrane. This study emphasises a major role of leak conductance in bacterial physiology and growth.