Glucose Concentration In Medium Research Articles

Microfluidic device flow field characterization around tumor spheroids with tunable necrosis produced in an optimized off-chip process.

Tumor spheroids are a 3-D tumor model that holds promise for testing cancer therapies in vitro using microfluidic devices. Tailoring the properties of a tumor spheroid is critical for evaluating therapies over a broad range of possible indications. Using human colon cancer cells (HCT-116), we demonstrate controlled tumor spheroid growth rates by varying the number of cells initially seeded into microwell chambers. The presence of a necrotic core in the spheroids could be controlled by changing the glucose concentration of the incubation medium. This manipulation had no effect on the size of the tumor spheroids or hypoxia in the spheroid core, which has been predicted by a mathematical model in computer simulations of spheroid growth. Control over the presence of a necrotic core while maintaining other physical parameters of the spheroid presents an opportunity to assess the impact of core necrosis on therapy efficacy. Using micro-particle imaging velocimetry (micro-PIV), we characterize the hydrodynamics and mass transport of nanoparticles in tumor spheroids in a microfluidic device. We observe a geometrical dependence on the flow rate experienced by the tumor spheroid in the device, such that the "front" of the spheroid experiences a higher flow velocity than the "back" of the spheroid. Using fluorescent nanoparticles, we demonstrate a heterogeneous accumulation of nanoparticles at the tumor interface that correlates with the observed flow velocities. The penetration depth of these nanoparticles into the tumor spheroid depends on nanoparticle diameter, consistent with reports in the literature.

The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms.

A yeast Saccharomyces cerevisiae Snf1 kinase, an analog of mammalian AMPK, regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.

Hyperglycemia and Hyperinsulinemia-Like Conditions Independently Induce Inflammatory Responses in Human Chondrocytes

To elucidate the mechanisms by which type 2 Diabetes Mellitus (DM2) constitutes a risk factor for the development and progression of osteoarthritis (OA), this work determined whether high glucose and/or high insulin, the hallmarks of DM2, are capable of activating the transcription factor, Nuclear Factor-κB (NF-κB), which plays a critical role in OA by inducing the expression of pro-inflammatory and catabolic genes. For this, we analyzed NF-κB activation by measuring the nuclear levels of p65 by western blot. As readouts of NF-κB activity, Interleukin-1β, Tumor Necrosis Factor-α, and inducible nitric oxide synthase (iNOS) expression were analyzed by real time RT-PCR and western blot. Culture of the human chondrocytic cell line, C28-I2, in high glucose (30 mM) increased nuclear NF-κB p65 levels in a time-dependent manner, relative to cells cultured in medium containing 10 mM glucose (regular culture medium). High glucose-induced NF-κB activation was inhibited by co-treatment with its specific inhibitor, Bay 11-7082, 5 µM. Culture of primary human chondrocytes under high glucose for 24 h increased IL-1β and TNF-α mRNA levels by 97% (p = 0.0066) and 85% (p = 0.0045), respectively, while iNOS mRNA and protein levels and NO production increased by 61% (p = 0.0017), 148% (p = 0.0089), and 70% (p = 0.049), respectively, relative to chondrocytes maintained in 10 mM glucose. Treatment of chondrocytic cells with 100 nM insulin was also sufficient to increase nuclear NF-κB p65 levels, independently of the glucose concentration in the culture medium. This study shows that hyperglycemia and hyperinsulinemia are independently sufficient to induce inflammatory responses in human chondrocytes, namely by activating NF-κB. This can be a relevant mechanism by which DM type 2 and other conditions associated with impaired glucose and insulin homeostasis, like obesity and the metabolic syndrome, contribute to the development and progression of OA.

Controlled swelling and in vitro release of insulin from konkoli grafted polymethylacrylaminde hydrogel

The extended study of konkoli grafted polymethylacrylaminde (KG-g-poly (MAAm) hydrogel synthesized in our earlier work from methacrylamide (MAAm) grafted and N,N-methylenebisacrylamide (N,N-MBAAm) crosslinked Konkoli ( Maesopsis eminni ) galactomannan (KG), is necessary to establish its potential application as an oral insulin delivery system. In this study, the kg-g-poly (MAAm) Hydrogel swelling in typical pH media show an initial rapid swelling before stabilizing. The hydrogel attains its parabolic peak swelling in distill water (pH = 7). Swelling rises from a lower pH medium (pH = 2.2), and in a medium with a slight pH above this (pH = 7.4), swelling starts dropping. Comparing the media for the insulin release studies, the cumulative release drops to its parabolic trough in the distill water medium (pH = 7). A higher cumulative release was recorded at a lower pH (pH = 2.2), and the highest cumulative release was recorded at a higher pH (pH = 7.4). The kinetics and mechanisms of these processes were presented while using the descriptive statistic of regression to fit the results from the processes to a power law. The release of insulin from kg-g-poly (MAAm) Hydrogel was also shown to increase with increase in glucose concentration in the media with an initial rapid release in all solutions, while in a typical intestinal pH media range, higher release at all concentrations was recorded at pH = 6.8.

Low glucose concentrations within typical industrial operating conditions have minimal effect on the transcriptome of recombinant CHO cells.

Typically, mammalian cell culture medium contains high glucose concentrations that are analogous to diabetic levels in humans, suggesting that mammalian cells are cultivated in excessive glucose. Using RNA-Seq, this study characterized the Chinese hamster ovary (CHO) cell transcriptome under two glucose concentrations to assess the genetic effects associated with metabolic pathways, in addition to other global responses. The initial extracellular glucose concentrations used represented high (30 mM) and low (10 mM) glucose conditions, where at the time the transcriptomes were compared, the glucose concentrations were approximately 24 and 4.4 mM for the mid-exponential cultures, where 4.4 mM represents a common target concentration in the biopharmaceutical industry for controlled fed-batch cultures. A recombinant CHO cell line producing a monoclonal antibody was used, such that the impact on glycosylation genes could be evaluated. Relatively few genes were identified as being significantly different (FDR ≤ 0.01) between the high and low glucose conditions, for example, only 575 genes, and only 40 of these genes had 2-fold or greater differences. Gene expression differences for glycolysis, TCA cycle, and glycosylation-related reactions were minimal and unlikely to have biological significance. This transcriptome study indicates that low glucose concentrations in the culture medium are unlikely to cause any biologically significant or detrimental changes to CHO cells at the transcriptome level. Furthermore, it is well-known that maintaining low glucose concentrations in fed-batch cultures can reduce lactate production, which in turn improves process outcomes. Taken together, the transcriptome data supports the continued development of low glucose-based processes to control lactate. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:771-785, 2017.

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233mg/L) far exceeded that by SHF (37.9mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

High-concentration glucose enhances invasion in invasive ductal breast carcinoma by promoting Glut1/MMP2/MMP9 axis expression.

Type 2 diabetes mellitus (T2DM) has been considered to be a risk factor for numerous human cancers. Hyperglycemia is one of the most direct internal environmental changes for patients with T2DM. Increasing evidence reveals that a high concentration of glucose can promote tumor progression, while its role for migration and invasion of invasive ductal breast carcinoma (IDBC) cells remains unclear. In the present study, it was demonstrated that IDBC patients with T2DM suffered an increased tumor size and more frequent lymphatic and distant metastasis compared with those without T2DM (P<0.05). MCF-7 breast carcinoma cells, which were cultured in a high glucose concentration medium (25.00 mM), exhibited increased invasion (P<0.05). In addition, the expression of glucose transporters (Gluts), matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in IDBC tissues with T2DM was significantly higher compared to those without T2DM. Downregulation of glucose transporter 1 (Glut1) by small interfering RNA may markedly suppress MCF-7 cell invasion as well as the expression of MMP2 and MMP9. These results suggest that T2DM can affect the malignant features of tumors in IDBC. The high glucose concentration in the tumor microenvironment may enhance IDBC invasion via upregulating Glut1/MMP2/MMP9 axis expression.

Air\u2013liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air–liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium–cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.

Development of a Ru Nanoparticle Loaded Thiol Functionalized Meso Porous Silica Modified Screen Printed Au Electrode for Electrochemical Detection and Estimation of Glucose.

Here we report the development of a glucose sensor based on electrochemical detection. The working electrode was a screen printed Au electrode, which was modified with Ru nanoparticle loaded thiol functionalized mesoporous silica. This sensor demonstrated its capability of detecting and estimating glucose concentration in aqueous medium over a wide range of concentration with high sensitivity, durability and reproducibility.

Regulation of PPARγ and CIDEC expression by adenovirus 36 in adipocyte differentiation.

This study is to investigate the role of adenovirus 36 (Ad36) in regulating expression of peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC) in Ad36-induced adipocyte differentiation. Human adipose-derived mesenchymal stem cells (hAMSCs) were isolated and cultured, and then infected with Ad36. Ad36-induced adipocytes were identified using quantitative real-time PCR and Oil red O staining. The expression levels of PPARγ and CIDEC in Ad36-induced adipocytes were determined by quantitative real-time PCR and Western blot analysis. Glucose uptake and intracellular triglyceride content were also determined in these induced cells. Our results from the Oil red O staining showed that Ad36 induced the differentiation of hAMSCs into human adipocytes in vitro. Moreover, the medium glucose concentration was significantly decreased, while the intracellular triglyceride content was significantly increased, in the Ad36-induced adipocytes, compared with the control group. Furthermore, our results showed that, the mRNA and protein expression levels of PPARγ and CIDEC were significantly upregulated in Ad36-induced adipocytes, in a time-dependent manner. On the other hand, compared with the control group, the CIDEC expression was downregulated when the Ad36-induced adipocytes were treated with the PPARγ inhibitor, GW9662. Ad36 could upregulate the expression level of CIDEC through increasing PPARγ expression during the adipocyte differentiation process.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Metabolic response of glioblastoma cells associated with glucose withdrawal and pyruvate substitution as revealed by GC-MS.

BackgroundTumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate.MethodsGlioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed.ResultsThe abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate.ConclusionsOur experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0131-9) contains supplementary material, which is available to authorized users.

Bioengineered human heparin with anticoagulant activity

Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.

Exobiopolymer production of Phytocordyceps sp. BCC 2744 and Akanthomyces pistillariiformis BCC 2694; optimization and scale-up

Production of exobiopolymers by Phytocordyceps sp. BCC 2744 and Akanthomyces pistillariiformis BCC 2694, interleukin-8 (IL-8) inducers, were optimized using experimental design. Biological and physiological properties of these exobiopolymers are attractive to use as wound-dressing material, so that it is very interesting to increase its production. Phytocordyceps sp. BCC 2744 and Akanthomyces pistillariiformis BCC 2694 were cultivated on different carbon and nitrogen sources and the best carbon and nitrogen sources for exobiopolymer production of Phytocordyceps sp. BCC 2744 were glucose and peptone, respectively, and 0.88 g/L exobiopolymer was obtained. Higher exobiopolymer (1.82 g/L) was obtained on glucose and meat extract by Akanthomyces pistillariiformis BCC 2694. After the effects of 4 variables were studied using a two-level fractional design, glucose and peptone concentration were the most influential parameters on exobiopolymer production of Phytocordyceps sp. BCC 2744. Lower exobiopolymer production was obtained in the medium supplemented with 5-Fluorouracil and vitamin solution at high level (10 mM and 3mL/L, respectively). The highest exobiopolymer production was obtained on 60 g/L glucose and 20 g/L peptone and 2.32 g/L exobiopolymer was produced. About 4.0 g/L exobiopolymer production in a 20 L bioreactor was obtained. At least 4 variables; medium type, nitrogen sources, glucose, and nitrogen concentration were applied on a two-level factorial design of biomass and exobiopolymer production of Akanthomyces pistillariiformis BCC 2694. Models obtained from the multiple regression are significant. At high level of glucose (60 g/L), phosphate medium, and 20 g/L meat extract, higher exobiopolymer production and about 2.54 g/L was obtained. About 4.5 g/L exobiopolymer production in 20 L bioreactor was obtained.

Abstract 32: Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3)

Abstract The tendency for enhanced glycolysis in cancer cells presents a target for chemotherapy. In previous studies we observed that proliferation of colon and bladder cancer cells can be inhibited by treatment with either phenformin or an inhibitor of PFKFB3 namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work we have examined the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15). The LDHA inhibitors that we chose to study in increasing order of IC50s were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) &amp;lt; isosafrole &amp;lt; oxamate. A synergistic anti-cancer effect of phenformin and oxamate has been reported. In the present work with colon and bladder cancer cells, additive but not synergistic growth inhibitory effects were seen with the LDHA inhibitors of which NHI-2 was effective at the lowest concentrations. Growth inhibition with PQP and PFK15 was compared in colon (Caco-2 and HT29) and bladder cancer cells (5637, HT1376, RT4, SW780, T24, TCCSUP and UM-UC-3). Apart from RT4 cells where the effects were similar, the effects were somewhat greater with PFK15 than with PQP and for both compounds the actions were seen at lower concentrations than in previous studies with 3PO. Actions on medium acidification and glucose uptake are more readily observed in the most rapidly growing cell lines. Effects of phenformin on medium pH and glucose concentration were decreased by the PFKFB3 and LDHA inhibitors that were examined. In accord with our previous studies on inhibitors of glycolysis, the increased medium acidification and glucose uptake caused by phenformin could be blocked by combined treatment with PFKFB3 or LDHA inhibitors. At the same time additive growth inhibitory effects were observed. The results supported the concept that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation while mitigating the lactic acidosis caused by phenformin as a single agent. Citation Format: Michael A. Lea, Yolanda Guzman, Charles desBordes. Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). [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 32.

P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture

Do high oxygen tension and high glucose concentrations dysregulate p66Shc (Src homologous-collagen homologue adaptor protein) expression during mouse preimplantation embryo culture? Compared with mouse blastocysts in vivo, P66Shc mRNA and protein levels in blastocysts maintained in vitro increased under high oxygen tension (21%), but not high glucose concentration. Growth in culture adversely impacts preimplantation embryo development and alters the expression levels of the oxidative stress adaptor protein p66Shc, but it is not known if p66Shc expression is linked to metabolic changes observed in cultured embryos. We used a standard wild-type CD1 mouse model of preimplantation embryo development and embryo culture with different atmospheric oxygen tension and glucose media concentrations. Changes to p66Shc expression in mouse blastocysts were measured using quantitative RT-PCR, immunoblotting and immunofluorescence followed by confocal microscopy. Changes to oxidative phosphorylation metabolism were measured by total ATP content and superoxide production. Statistical analyses were performed on a minimum of three experimental replicates using Students' t-test or one-way ANOVA. P66Shc is basally expressed during in vivo mouse preimplantation development. Within in vivo blastocysts, p66Shc is primarily localized to the cell periphery of the trophectoderm. Blastocysts cultured under atmospheric oxygen levels have significantly increased p66Shc mRNA transcript and protein abundances compared to in vivo controls (P<0.05). However, the ratio of phosphorylated serine 36 (S36) p66Shc to total p66Shc decreased in culture regardless of O2 atmosphere used, supporting a shift in the mitochondrial fraction of p66Shc. Total p66Shc localized to the cell periphery of the blastocyst trophectoderm and phosphorylated S36 p66Shc displayed nuclear and cytoplasmic immunoreactivity, suggesting distinct compartmentalization of phosphorylated S36 p66Shc and the remaining p66Shc fraction. Glucose concentration in the culture medium did not significantly change p66Shc mRNA or protein abundance or its localization. Blastocysts cultured under low or high oxygen conditions exhibited significantly decreased cellular ATP and increased superoxide production compared to in vivo derived embryos (P<0.05). This study associates embryonic p66Shc expression levels with metabolic abnormalities but does not directly implicate p66Shc in metabolic changes. Additionally, we used one formulation of embryo culture medium that differs from that used in other mouse model studies and from clinical media used to support human blastocyst development. Our findings may, therefore, be limited to this media, or may be a species-specific phenomenon. This is the first study to show distinct immunolocalization of p66Shc to the trophectoderm of mouse blastocysts and that its levels are abnormally increased in embryos exposed to culture conditions. Changes in p66Shc expression and/or localization could possibly serve as a molecular marker of embryo viability for clinical applications. The outcomes provide insight into the potential metabolic role of p66Shc. Metabolic anomalies are induced even under the current optimal culture conditions, which could negatively impact trophectoderm and placental development. Not applicable. Canadian Institutes of Health Research (CIHR) operating funds, Ontario Graduate Scholarship (OGS). There are no competing interests.

A Continuously Infused Microfluidic Radioassay System for the Characterization of Cellular Pharmacokinetics.

The CIMR system consists of a microfluidic chip integrated with a flow-control unit and a positron camera. Medium diluted with radioactive tracer flows through a cell chamber continuously at low speed. Positrons emitted from the cells and from tracer in the medium are measured with the positron camera. The human cell lines SkBr3 and Capan-1 were incubated with media of 3 different glucose concentrations and then measured with 18F-FDG on the CIMR system. In addition, a conventional uptake experiment was performed. The relative uptake ratios between different medium conditions were compared. A cellular 2-compartment model was applied to estimate the cellular pharmacokinetics on CIMR data. The estimated pharmacokinetic parameters were compared with expressions of glucose transporter-1 (GLUT1) and hexokinase-2 measured by quantitative real-time polymerase chain reaction. The relative uptake ratios obtained from CIMR measurements correlated significantly with those from the conventional uptake experiments. The relative SDs of the relative uptake ratios obtained from the CIMR uptake experiments were significantly lower than those from the conventional uptake experiments. The fit of the cellular 2-compartment model to the 18F-FDG CIMR measurements was of high quality. For SkBr3, the estimated pharmacokinetic parameters k1 and k3 were consistent with the messenger RNA expression of GLUT1 and hexokinase-2: culturing with low glucose concentrations led to higher GLUT1 and hexokinase-2 expression as well as higher estimated k1 and k3 For Capan-1, the estimated k1 and k3 increased as the glucose concentration in the culture medium decreased, and this finding did not match the corresponding messenger RNA expression. The CIMR system captures dynamic uptake within the cell culture and enables estimation of the cellular pharmacokinetics.

Zuogui Wan rescues the high-glucose-induced damaging effects on early embryo development

BackgroundHigh concentration of glucose in culture medium affects the developmental process and the quality of the pre-implantation embryo. This study examined the effects of Zuogui Wan (ZGW) supplementation on early embryo development cultured in high-glucose medium.MethodsEmbryos were cultured in high-glucose medium with or without ZGW supplementation. Developmental rate and competence was evaluated by cleavage rate, blastocyst rate, and blastocyst total cell number, reactive oxygen species (ROS) level, glutathione (GSH) concentration, and metabolome were also measured to determine the effect of ZGW on embryo development at the cellular level.ResultsCompared with the vehicle group, supplementation of 0.01 % (v/v) ZGW to high-glucose medium significantly increased cleavage rate (80.1 ± 1.0 % vs 72.1 ± 1.3 %), blastocyst rate (50.5 ± 1.0 % vs 41.3 ± 1.7 %), and blastocyst total cell number (63.2 ± 2.2 vs 57.2 ± 1.6). ROS level was lower and GSH concentration in blastocysts was higher in ZGW-treated group. Metabolomic analysis found that the ratio of glucose to succinic acid and glucose to fumaric acid were lower in the ZGW-treated group .ConclusionsDevelopmental rates of zygotes in high-glucose culture medium were significantly lower than those in regular culture medium. ZGW supplementation significantly improved embryo development and quality in high-glucose medium. Supplementing ZGW in high-glucose medium also significantly increased total cell number and GSH concentration but decreased ROS level in blastocysts likely by modifying metabolic profile during embryo development. Together, these data suggest that supplementation of ZGW rescues high-glucose-induced detrimental effects on pre-implantation embryo development.

The role of N-glycosylation in high glucose-induced upregulation of intercellular adhesion molecule-1 on bovine retinal endothelial cells.

The development of diabetic retinopathy has been implicated as a consequence of chronic inflammation. Given the role of the intercellular adhesion molecule-1 (ICAM-1) in inflammation, the potential effect of N-glycosylation on the upregulated expression of ICAM-1 at the surface of bovine retinal endothelial cells (BRECs) induced by high glucose concentrations was investigated. Gene and protein expression of ICAM-1 in primary BRECs cultured in medium containing increasing concentrations of mannose or glucose in the presence or absence of tunicamycin were studied with reverse transcription-polymerase chain reaction and Western blot analysis, and the expression level of ICAM-1 at the surface of BRECs was examined with an immunofluorescence analysis. A lectin blot assay with PHA-L was performed to explore the level of N-glycans on cell total proteins or immunoprecipitated ICAM-1 from cells treated or untreated with high glucose. Both the mRNA and protein levels of ICAM-1, as well as the level of ICAM-1 on the cell surface, were significantly upregulated by increasing the concentration of glucose in the culture medium, with a peak concentration of 20mm. Consistent with these results, a dramatic increase in the N-glycosylation of ICAM-1 in BRECs cultured with a high concentration of glucose was observed, which could be partially attenuated by tunicamycin treatment. High glucose-induced upregulation of ICAM-1 on the surface of BRECs could be ascribed to the alterations in its N-glycosylation at least in part, indicating that interference with the glycosylation of ICAM-1 may contribute to improving the efficiency of current therapies with diabetic retinopathy.

Nitrogen and carbon source balance determines longevity, independently of fermentative or respiratory metabolism in the yeast Saccharomyces cerevisiae.

Dietary regimens have proven to delay aging and age-associated diseases in several eukaryotic model organisms but the input of nutritional balance to longevity regulation is still poorly understood. Here, we present data on the role of single carbon and nitrogen sources and their interplay in yeast longevity. Data demonstrate that ammonium, a rich nitrogen source, decreases chronological life span (CLS) of the prototrophic Saccharomyces cerevisiae strain PYCC 4072 in a concentration-dependent manner and, accordingly, that CLS can be extended through ammonium restriction, even in conditions of initial glucose abundance. We further show that CLS extension depends on initial ammonium and glucose concentrations in the growth medium, as long as other nutrients are not limiting. Glutamine, another rich nitrogen source, induced CLS shortening similarly to ammonium, but this effect was not observed with the poor nitrogen source urea. Ammonium decreased yeast CLS independently of the metabolic process activated during aging, either respiration or fermentation, and induced replication stress inhibiting a proper cell cycle arrest in G0/G1 phase. The present results shade new light on the nutritional equilibrium as a key factor on cell longevity and may contribute for the definition of interventions to promote life span and healthy aging.