Metabolic State Of Cell Research Articles

Nicotinamide adenine dinucleotide induces a bivalent metabolism and maintains pluripotency in human embryonic stem cells.

Nicotinamide adenine dinucleotide (NAD+ ) and its precursor metabolites are emerging as important regulators of both cell metabolism and cell state. Interestingly, the role of NAD+ in human embryonic stem cell (hESC) metabolism and the regulation of pluripotent cell state is unresolved. Here we show that NAD+ simultaneously increases hESC mitochondrial oxidative metabolism and partially suppresses glycolysis and stimulates amino acid turnover, doubling the consumption of glutamine. Concurrent with this metabolic remodeling, NAD+ increases hESC pluripotent marker expression and proliferation, inhibits BMP4-induced differentiation and reduces global histone 3 lysine 27 trimethylation, plausibly inducing an intermediate naïve-to-primed bivalent metabolism and pluripotent state. Furthermore, maintenance of NAD+ recycling via malate aspartate shuttle activity is identified as an absolute requirement for hESC self-renewal, responsible for 80% of the oxidative capacity of hESC mitochondria. Our findings implicate NAD+ in the regulation of cell state, suggesting that the hESC pluripotent state is dependent upon cellular NAD+ .

Patch-Clamp Fluorometry Defines a Role for SUR1 in Nucleotide Inhibition of KATP Channels

ATP sensitive K+ (KATP) channels couple a cell's metabolic state to its electrical excitability. KATP is a hetero-octameric complex, with four pore-forming inward rectifier K+ channel subunits (Kir6.1 or Kir6.2), each associated with a modulatory sulfonylurea receptor (SUR1 or SUR2A/B). ATP/ADP binding to a site on the Kir6.2 subunit shuts KATP. In the presence of Mg2+, nucleotide binding to SUR1 stimulates KATP. However, SUR1 also has direct effects on gating at Kir6.2 in the absence of Mg2+. The presence of SUR1 increases channel open probability, but paradoxically also increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We introduced the fluorescent non-canonical amino acid ANAP at a position (W311ANAP) in the cytoplasmic C-terminal region of Kir6.2 co-expressed with SUR1 in HEK 293T cells. This allowed us to simultaneously measure channel currents and inhibitory nucleotide binding to Kir6.2 via FRET between ANAP and a fluorescent ATP derivative (TNP-ATP). We fit models to the combined data sets to produce well-constrained estimates of open-state nucleotide affinity and the strength of coupling between nucleotide binding and channel closure. Our models suggest that “wild-type” KATP is closed when only one of its nucleotide binding sites is occupied. A Kir6.2 mutation (C166S) that profoundly increases channel activity had very little effect on nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. In contrast, mutations at position E205 on SUR1, which is located on an intracellular loop close to the inhibitory site on Kir6.2, affected both TNP-ATP affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in contributing to nucleotide binding to the open state and in stabilizing the nucleotide-bound closed state.

Fiber Diameter Differentially Regulates Function of Retinal Pigment and Corneal Epithelial Cells on Nanofibrous Tissue Scaffolds.

Biomaterials have significant functions as tissue scaffolds to support cells for regeneration. Nanofibrous scaffolds which mimic the architecture of the extracellular matrix are well suited to support epithelial cells for ocular tissue engineering. This study aimed at investigating the role of scaffold architecture, if any, on the response of ocular epithelial cells. Thus, we have cultured two different types of ocular epithelial cells on nanofibrous scaffolds of two different diameters to evaluate their generic and cell-specific properties. Human adult retinal pigment epithelial (ARPE-19) and human corneal epithelial (HCE-T) cells were cultured on poly(ε-caprolactone) (PCL) nanofibers of different diameters, nominally 500 and 1300 nm. Moduli of the fiber mats were marginally different at 7.4 and 11.1 kPa for 500 and 1300 nm diameter, respectively. The molecular changes in the cells in response to the different fibers were analyzed by qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, MTT assay, and SEM to assess properties such as proliferation, apoptosis, membrane potential, epithelial-mesenchymal transition, stem cell population, VEGF-A secretion, differentiation, and metabolic status of the cells. HCE-T cells revealed characteristic morphology along with higher expression of proliferation, differentiation, and lower apoptotic markers when cultured on PCL nanofibers of 500 nm. However, on nanofibers of 1300 nm, the cells showed higher expression of the corneal stem/progenitor as well as pluripotent stem cell markers. ARPE-19 cells exhibited characteristic hexagonal morphology with elevated expression levels of proliferative markers, phagocytic activity, and lower apoptosis levels. However, on 500 nm nanofibers, they expressed higher levels of pluripotent markers and secretion of VEGF-A. These findings demonstrate that the response can differ markedly from scaffold architecture even if derived from the same tissue and originating from the same germ layer. Furthermore, it paves the way for a target specific outcome and, thereby, for personalized translational medicine.

Role of Sirtuins in Tumor Angiogenesis.

Generally, changes in the metabolic status of cells under conditions like hypoxia and accumulation of lactate can be sensed by various sensing mechanisms, leading to modulation of a number of signal transduction pathways and transcription factors. Several of the proangiogenic cytokines like VEGF, FGF, PDGF, TGF-β, Ang-2, ILs, etc. are secreted by cancer cells, under hypoxic microenvironment. These cytokines bind to their receptors on the endothelial cells and activates a number of signaling pathways including Akt/PIP3, Src, p38/MAPK, Smad2/3, etc., which ultimately results in the proliferation and migration of endothelial cells. Transcription factors that are activated in response to the metabolic status of tumors include HIFs, NF-κb, p53, El-2, and FOXO. Many of these transcription factors has been reported to be regulated by a class of histone deacetylase called sirtuins. Sirtuins are NAD+ dependent histone deacetylases that play pivotal role in the regulation of tumor cell metabolism, proliferation, migration and angiogenesis. The major function of sirtuins include, deacetylation of histones as well as some non-histone proteins like NF-κB, FOXOs, PPAR⋎, PGC1-α, enzymes like acetyl coenzymeA and structural proteins like α tubulin. In the cell, sirtuins are generally considered as the redox sensors and their activities are dependent on the metabolic status of the cell. Understanding the intricate regulatory mechanisms adopted by sirtuins, is crucial in devising effective therapeutic strategies against angiogenesis, metastasis and tumor progression. Keeping this in mind, the present review focuses on the role of sirtuins in the process of tumor angiogenesis and the regulatory mechanisms employed by them.

Phasor analysis of fluorescence lifetime data and its application

Fluorescence lifetime imaging microscopy (FLIM) is widely used in biomedical, materials and other fields. It not only has strong specificity and high sensitivity, but also has the capability of quantitative measurement because the fluorescence lifetime is not affected by the intensity of excitation, the concentration of fluorophores and photobleaching, and consequently is able to monitor the changes of microenvironment and reflecting the interaction between molecules. However, its application is limited to some extent by the complexity of data analysis. In order to make FLIM technology more suitable for fast analysis of high-throughput data, a variety of new algorithms for fluorescence lifetime analysis have emerged in recent years, such as phasor analysis, maximum likelihood estimation, first-order moment, Bayesian analysis, and compressed sensing. Among them, the phasor analysis (PA) method obtains the fluorescence lifetime by converting the fitting in the time domain to the direct calculation in the frequency domain. Compared with traditional least-square fitting method, it is not only simpler and faster, but also more suitable for the case of low photon counts. In addition, in the PA approach to FLIM, the fluorescence decay is directly converted into a phasor diagram by simple mathematics, where the phasor points originating from different pixels in the image are represented by the positions in the phasor plot, and thus the graphical representation obtained by PA method is convenient for data visualization and cluster analysis. Therefore, it has become a simple and powerful analysis method for FLIM, and is increasingly favored by researchers. In this paper, the basic principle of PA method and how we can use it are described in detail. And on this basis, the latest application research progress of the method in cell metabolism state measurement, protein interaction study, cell microenvironment measurement, auxiliary pathological diagnosis, and resolution improvement in super-resolution imaging are introduced and summarized. The advantages of PA method in these FLIM applications are focused on, providing useful reference for the research in related fields. Finally, the phasor analysis method for FLIM data analysis and the development trend of its application are prospected.

INFLUENCE OF EXOGENOUS GIBBERELLIN AND ITS MIXTURE WITH SUCROSE ON THE FORMATION OF XYLEM INCREMENT OF SILVER BIRCH

We studied the effect of gibberellin on cambial activity towards the xylem formation in adult birch trees (Betula pendula Roth var. pendula, common birch). The injection of exogenous solutions was used as a methodological technique. To do this, “chambers” were made on tree trunks by cutting out the outer layers of the bark up to the inner parts of the non-conductive phloem, leaving the conductive phloem and cambial zone intact. GA3 gibberellin solutions (0.005%, 0.01%, 0.02%, 0.05%, 0.1%, 0.2 %) and a mixture of gibberellin with sucrose (GA3 0.01% + sucrose 1% - 20%) were injected into the chambers using a syringe. A strong stimulating effect of the hormone on the formation of xylem increment has been established. It was not possible to identify the dependence of the growth increment on the concentrations used, since their values obviously exceeded the level at which the maximum possible response of the cambial zone to the action of gibberellin was achieved with a given metabolic status of cells and tissues. This is confirmed by the effect of the residual action of exogenous solutions, which manifested itself after the termination of their administration: in variants with higher concentrations of the hormone, the final increase in xylem was greater. It was demonstrated for the first time that high sucrose concentrations (10% and 20%) reduce the stimulating effect of gibberellin. It has been suggested that in this case, sucrose can act through an increase in the level of UDP-glucose, the interaction of which with gibberellin leads to its conjugation (inactivation). The reaction of UDP-glucose with auxin cannot also be excluded, as a result of which it’s the conjugate IAA-glucose is formed, since gibberellin provides acceleration of cambial growth only in the presence of physiologically active (free) auxin in the tissue.

Macrophage Function in the Pathogenesis of Non-alcoholic Fatty Liver Disease: The Mac Attack.

Obesity is a prevalent predisposing factor to non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease in the developed world. NAFLD spectrum of disease involves progression from steatosis (NAFL), to steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Despite clinical and public health significance, current FDA approved therapies for NAFLD are lacking in part due to insufficient understanding of pathogenic mechanisms driving disease progression. The etiology of NAFLD is multifactorial. The induction of both systemic and tissue inflammation consequential of skewed immune cell metabolic state, polarization, tissue recruitment, and activation are central to NAFLD progression. Here, we review the current understanding of the above stated cellular and molecular processes that govern macrophage contribution to NAFLD pathogenesis and how adipose tissue and liver crosstalk modulates macrophage function. Notably, the manipulation of such events may lead to the development of new therapies for NAFLD.

Effects of Me2SO and Trehalose on the Cell Viability, Proliferation, and Bcl-2 Family Gene (BCL-2, BAX, and BAD) Expression in Cryopreserved Human Breast Cancer Cells.

Long-term cryopreservation of the viability and metabolic state of cells in cancer cell/tissue specimens has significant implications for diagnostic verification of disease progression in cancer patients and selection of effective treatment options via development of the patient-derived xenograft (PDX) models for drug screening. The purpose of this study is to investigate the effects of cryoprotectant agents (CPAs) on the expression of BCL-2 family genes (BCL-2, BAX, and BAD) that are involved in the growth and development of breast cancers. MCF-7 cells were cryopreserved in Dulbecco's modified Eagle's medium (DMEM) with 20% (v/v) fetal bovine serum, using 10% (v/v) Me2SO (dimethyl sulfoxide, DMSO) or 7.5% (v/v) Me2SO with 100is-300 mM trehalose as cryoprotectant solutions. After storage at -80°C for 7 days, the cells were thawed for evaluation. The use of Me2SO and trehalose has affected cell survival, proliferation, apoptotic state, as well as BCL-2 family gene expression. The conventional 10% (v/v) Me2SO method yields ∼80% post-thaw cell survival and good cell proliferation, but it drastically alters the pattern of the BCL-2 family gene expression. The antiapoptotic gene BCL-2 is downregulated, whereas two proapoptotic genes BAX and BAD are upregulated. The partial substitution of Me2SO with 200 or 300 mM trehalose enhances cell proliferation of survived cells after cryopreservation. The presence of trehalose upregulates the expression of both the antiapoptotic gene BCL-2 and proapoptotic genes BAX and BAD. Cryopreservation could tip off the checkpoint of the apoptotic pathway regulated by the BCL-2 family members, and the effect may be protectant dependent. The findings of this study demonstrate the importance of paying attention to the potential change of gene expression and metabolic state of cancer cells after cryopreservation in an attempt to development of the PDX models from cryopreserved cancer cells or tissue specimens.

Resveratrol Improves Boar Sperm Quality via 5'AMP-Activated Protein Kinase Activation during Cryopreservation.

Mammalian sperm is highly susceptible to the reactive oxygen species (ROS) stress caused by biochemical and physical modifications during the cryopreservation process. 5′AMP-activated protein kinase (AMPK) is involved in regulating both cell metabolism and cellular redox status. The aim of the present study was to investigate whether the resveratrol protects boar sperm against ROS stress via activation of AMPK during cryopreservation. Boar sperm was diluted with the freezing medium supplemented with resveratrol at different concentrations (0, 25, 50, 75, 100, and 125 μM). It was observed that the addition of 50 μM resveratrol significantly improved the postthaw sperm progressive motility, membrane integrity, acrosome integrity, mitochondrial activity, glutathione (GSH) level, activities of enzymatic antioxidants (glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase), and the phosphorylation of AMPK. Meanwhile, the lipid peroxidation, ROS levels, and apoptosis of postthaw sperm were reduced in the presence of 50 μM resveratrol. Furthermore, when fresh boar sperm was incubated with the medium in the presence of 50 μM resveratrol and 30 μM Compound C (an AMPK inhibitor), the effects of the resveratrol were partly counteracted by the Compound C. These observations suggest that the resveratrol protects boar sperm via promoting AMPK phosphorylation. In conclusion, the addition of resveratrol to the freezing extenders protects boar sperm against ROS damage via promoting AMPK phosphorylation for decreasing the ROS production and improving the antioxidative defense system of postthaw sperm. These findings provide novel insights into understanding the mechanisms of resveratrol on how to protect boar sperm quality contrary to the ROS production during cryopreservation.

Abstract 4142: Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells

Abstract The role of the PI3K/mTOR and MAPK signaling pathways in regulating T cell activation and differentiation is well established. It has been shown that mTOR acts as a sensor of the T cell metabolic state, coordinating diverse inputs to determine the balance between effector versus memory cell fates. Similarly, the MAPK interacting kinases, MNK1 and MNK2, are key downstream effector kinases that mediate post-transcriptional gene regulation of critical mediators of the T cell response, including immune checkpoint proteins and cytokines, via the phosphorylation of specific RNA binding proteins such as eIF4E and hnRNPA1. eFT508 is a potent and selective inhibitor of both MNK1 and MNK2, which enhances anti-tumor immune responses by decreasing the expression of immune response modulators including PD-1, PD-L1, LAG3, TIM3 and IL-10. Furthermore, eFT508 treatment boosts memory T cell populations while increasing the effectiveness of cytotoxic T lymphocytes. Phosphoproteomic analysis of the effects of eFT508 on early T cell activation identified novel eFT508 sensitive phosphopeptides, including a subset that overlaps with mTOR-dependent phosphorylation sites. In vitro biochemical analysis has shown that eFT508 is not a mTOR kinase inhibitor and that most of these sites are not direct MNK substrates. These results suggest inhibition of MNK by eFT508 can have significant effects on mTOR signaling. Consistent with these findings, we observe a physical association between MNK and mTOR that is disrupted by either eFT508 or the allosteric mTOR inhibitor rapamycin. Our phosphoproteomic analysis also identified novel MNK-dependent phosphorylation sites within the translation initiation factor eIF4G1 that regulate the ability of mTOR to recognize eIF4G1 as a substrate. In addition, phosphorylation of the mTOR target 4EBP1 is decreased upon treatment of T cells with eFT508, although 4EBP1 itself is not a direct substrate of MNK in vitro. These data are consistent with a role for MNK in regulating mRNA translation, not only through phosphorylation of eIF4E, but also by modulating the activity of mTOR toward specific substrates. Given the role of mTOR in controlling memory T cell differentiation, our results provide a potential mechanistic basis to explain the impact of MNK inhibition on T cell differentiation. Ongoing studies are further characterizing the consequences of eFT508 regulation on MNK/mTOR signaling and T cell differentiation. This work significantly expands our current understanding of eFT508’s mechanism of action as well as its ability to promote a prolonged anti-tumor immune response. Citation Format: Craig R. Stumpf, Vikas K. Goel, Rajesh K. Sharma, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4142.

Abstract 1201: IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer

Abstract IL-4 has a number of well-established functions in immune cells including proliferation, and survival of lymphocytes, polarization of macrophages to the pro-tumor M2 tumor associated macrophage phenotype. Another role of IL-4 signaling is induction of metabolic changes in immune cells including increased glycolysis in B-lymphocytes, and increased expression of arginase 1 with a switch to fatty acid oxidation in macrophages. However, the effects of IL-4 signaling in tumor cells that express IL-4 receptor is not well understood. Previous work from our lab has shown that loss of IL-4 receptor (IL-4R) significantly abrogates metastasis in experimental metastasis models in mice. Thus, we wondered whether changes in metabolism downstream of IL-4 receptor activation may contribute to metastatic phenotypes. Since altered metabolic pathways have been shown to regulate the epigenetic state of cancer cells, we are pursuing histone modifications as a mechanism for the connection between altered metabolism and increased metastatic capability in IL-4R expressing breast cancer cells. MDA-MB-231 and BT549 triple negative breast cancer cells were treated with IL-4 for 2,4,6, and 8 days. Samples of media were assayed for glutamine and glucose consumption, as well as lactate and glutamate secretion. IL-4 decreased consumption of glucose and glutamine, yet concomitantly increased the release of lactate and glutamate. These data suggest that IL-4 alters the utilization of glucose and glutamine in cancer cells. To study potential links to epigenetic regulation cells were treated for 18, or 48 hours with IL-4 before biochemical fractionation was performed in order to obtain nuclear and cytosolic lysates. The nuclear fractions were assessed for histone acetyltransferase (HAT) activity using commercially available assays, and showed that IL-4 increased the activity of histone acetyltransferases. We also performed pull down of acetylated lysine residue from the nuclear fractions of IL-4 treated cells. The cells were then assessed for abundance of acetylated histone 3. In addition, we used western blotting to measure the abundance of specific histone modifications such as histone 3 lysine 9 acetylation and histone 3 lysine 27 acetylation. Overall, histone acetyltransferase activity and levels of histone acetylation were increased by IL-4. Additionally, we found that IL4 increases the acetylation of histone 3 lysine 9. In order to determine the effects of IL-4 signaling on metabolism in vivo we have initiated PET imaging studies in humanized mice that underwent tail vein injections of BT549 cell lines. These mice were treated with MDNA413, an antagonist of IL-4 signaling, and are being assessed for uptake of glucose and glutamine. Together these suggest that IL-4 alters the metabolism and epigenetic state of cancer cells and may be a viable target for therapy in metastatic breast cancer. Citation Format: Demond Williams, Adam Miranda, Chilesi Uririri, Barbara Fingleton. IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1201.

Effect of Curcumin Supplement on Pulmonary Functions, Total and Differential White Blood Cell Count, Serum Level of Leptin and Body Mass Index in a Sample of Iraqi Patients with Chronic Bronchial Asthma

Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation together with increased oxidative stress that lead to clinical symptoms of asthma. 
 Obesity is a known risk factor of asthma as the obesity is correlated with systemic inflammation and airway restriction. Curcumin, a natural product derived from the rhizomes of Curcuma longa (Turmeric), has a wide range of beneficial properties including anti-inflammatory and weight reducing agent. This study was designed to evaluate the effect of curcumin as supplement therapy on the pulmonary function, total and differential white blood cell count and metabolic status of chronic bronchial asthmatic patients. It is a prospective randomized controlled interventional study carried out on 40 patients visiting Al-Yarmouk teaching hospital and conducted from October 2017 to April 2018. The patients were allocated into group1; include 17 asthmatic patients assigned to receive conventional therapy for asthma alone, and group 2; include 23 asthmatic patients assigned to receive conventional therapy for asthma plus 750 mg curcumin capsule twice daily for two months. Results revealed significant improvement in forced expiratory volume in one second (FEV1) and the ratio of forced expiratory volume in one second to forced vital capacity (FEV1/FVC) in group 2 patients after two months(P<0.01). The total white blood cell count didn’t show any difference after treatment except decrease in neutrophil count after curcumin supplement (p< 0.05). The leptin level and body mass index present with no significant difference in both groups after two months (p >0.05).

Mitochondrial Dysfunction and Diabetes: Is Mitochondrial Transfer a Friend or Foe?

Obesity, insulin resistance and type 2 diabetes are accompanied by a variety of systemic and tissue-specific metabolic defects, including inflammation, oxidative and endoplasmic reticulum stress, lipotoxicity, and mitochondrial dysfunction. Over the past 30 years, association studies and genetic manipulations, as well as lifestyle and pharmacological invention studies, have reported contrasting findings on the presence or physiological importance of mitochondrial dysfunction in the context of obesity and insulin resistance. It is still unclear if targeting mitochondrial function is a feasible therapeutic approach for the treatment of insulin resistance and glucose homeostasis. Interestingly, recent studies suggest that intact mitochondria, mitochondrial DNA, or other mitochondrial factors (proteins, lipids, miRNA) are found in the circulation, and that metabolic tissues secrete exosomes containing mitochondrial cargo. While this phenomenon has been investigated primarily in the context of cancer and a variety of inflammatory states, little is known about the importance of exosomal mitochondrial transfer in obesity and diabetes. We will discuss recent evidence suggesting that (1) tissues with mitochondrial dysfunction shed their mitochondria within exosomes, and that these exosomes impair the recipient’s cell metabolic status, and that on the other hand, (2) physiologically healthy tissues can shed mitochondria to improve the metabolic status of recipient cells. In this context the determination of whether mitochondrial transfer in obesity and diabetes is a friend or foe requires further studies.

Effect of Curcumin Supplement on Pulmonary Functions, Total and Differential White Blood Cell Count, Serum Level of Leptin and Body Mass Index in a Sample of Iraqi Patients with Chronic Bronchial Asthma

Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation together with increased oxidative stress that lead to clinical symptoms of asthma. Obesity is a known risk factor of asthma as the obesity is correlated with systemic inflammation and airway restriction. Curcumin, a natural product derived from the rhizomes of Curcuma longa (Turmeric), has a wide range of beneficial properties including anti-inflammatory and weight reducing agent. This study was designed to evaluate the effect of curcumin as supplement therapy on the pulmonary function, total and differential white blood cell count and metabolic status of chronic bronchial asthmatic patients. It is a prospective randomized controlled interventional study carried out on 40 patients visiting Al-Yarmouk teaching hospital and conducted from October 2017 to April 2018. The patients were allocated into group1; include 17 asthmatic patients assigned to receive conventional therapy for asthma alone, and group 2; include 23 asthmatic patients assigned to receive conventional therapy for asthma plus 750 mg curcumin capsule twice daily for two months. Results revealed significant improvement in forced expiratory volume in one second (FEV1) and the ratio of forced expiratory volume in one second to forced vital capacity (FEV1/FVC) in group 2 patients after two months(P<0.01). The total white blood cell count didn’t show any difference after treatment except decrease in neutrophil count after curcumin supplement (p< 0.05). The leptin level and body mass index present with no significant difference in both groups after two months (p >0.05).

Label-free spectroscopy for cell monitoring

Background & Aim Cell monitoring is essential to the quality assurance and control of cell-based products, especially those used in therapeutics. Conventionally, sampling is performed at various stages of the manufacturing process to probe for the condition of cells in terms of viability, potency, phenotype and other characteristics. This is achieved by either an indirect assessment of the cells’ health through measurements of contents in their environment or a direct but destructive measurement of the cells. Indirect measurements are not absolute and require reference either to earlier time points or a calibration curve, while destructive testing is not ideal as it results in wastage and increases the risk of contamination. Considering that cells are the product in cell-based therapeutics, it is necessary that direct measurements be made to access their quality. The high cost of such therapeutics further motivates the development of non-destructive measurement technologies, so as to maximize yield. Methods, Results & Conclusion We developed a method of monitoring that is able to directly measure cell characteristics without sacrificing cell samples. The method is based on optical spectroscopy, where the collected signal is distributed according to its wavelength in a spectrum. Specifically, we made use of cell autofluorescence, which is the emission from native metabolic coenzymes when excited by ultraviolet A light. We studied the autofluorescence spectra for cultured cells at different levels of confluency, from 20% to 90% confluence in over 450 spectra. Spectral data were first signal processed and analysed using a spectral decomposition method, from which a redox ratio was computed. The redox ratio is a representative measurement of the metabolic state of cells. A strong correlation between cell confluence and redox ratio was noted, with statistical significance observed at 20% confluence intervals. In order to implement this correlation in monitoring, machine learning was applied to raw spectral data to train classification and regression models with supervision. The trained models achieved accuracies of up to 70% when predicting cell confluences from autofluorescence spectra. Following our measurements, assays were conducted and results validate that ultraviolet A light did not adversely affect cell viability and DNA integrity. In conclusion, we demonstrated the use of a label-free and non-destructive method in determining cell metabolic states with correlation to cell confluence.

Effects of gold and PCL- or PLLA-coated silica nanoparticles on brain endothelial cells and the blood-brain barrier.

Nanomedicine is a constantly expanding field, facilitating and improving diagnosis and treatment of diseases. As nanomaterials are foreign objects, careful evaluation of their toxicological and functional aspects prior to medical application is imperative. In this study, we aimed to determine the effects of gold and polymer-coated silica nanoparticles used in laser tissue soldering on brain endothelial cells and the blood–brain barrier using rat brain capillary endothelial cells (rBCEC4). All types of nanoparticles were taken up time-dependently by the rBCEC4 cells, albeit to a different extent, causing a time- and concentration-dependent decrease in cell viability. Nanoparticle exposure did not change cell proliferation, differentiation, nor did it induce inflammation. rBCEC4 cells showed blood–brain barrier characteristics including tight junctions. None of the nanoparticles altered the expression of tight junctions or impaired the blood–brain barrier permeability. The findings suggest that effects of these nanoparticles on the metabolic state of cells have to be further characterized before use for medical purposes.

OR12-2 Obesity Induces Changes in T Cell Metabolism and Function

Low-grade inflammation is responsible for many obesity-associated pathologies such as insulin resistance, type 2 diabetes and reduction in protective immunity. T cells play an important role in mounting an effective immune response and the modulation of inflammation, and have been identified as a key player in obesity-associated inflammation. Altered T cell metabolism can influence T cell differentiation and function; however, it is not yet known if or how obesity influences the T cell metabolic state. We hypothesized that diet induced obesity promotes changes in T cell metabolism leading to increased pro-inflammatory T cells function. To test our hypothesis, we assessed the effect of diet-induced obesity (DIO) on T cell subpopulation number and metabolism in the visceral adipose tissue (VAT) and peripheral lymphoid tissue (spleen). To achieve that, C57BL/6 mice were placed on high fat (DIO mice) or regular chow diet (control mice) at weaning and remained on diet for 18 weeks. VAT and spleen were collected and T cells were isolated and analyzed by flow cytometry for: T cells subpopulation markers, glucose uptake, and cytokine production. In addition, the expression of almost 240 enzymes involved in several metabolic pathways were measured in CD4+ T cells by RT-qPCR. We found that proportions of Treg cells were decreased in the VAT of DIO mice while CD8+ T cells increased, indicating an inflammatory environment in the adipose tissue of the DIO mice. CD4+ T cells isolated from the spleen of DIO mice were also found to be metabolically altered with increased glucose uptake and elevated oxygen consumption rate (OCR; a marker of mitochondrial metabolism). The spare respiratory capacity, an indicator of mitochondrial reserve, of CD4+ T cells from DIO mice was found to be reduced by the fatty acids oxidation inhibitor Etomoxir, while the pyruvate transport inhibitor UK5099 was found to have no significant effect, indicating a preference toward the use of fatty acids as a mitochondrial fuel source. The metabolic qPCR panel showed slight differences in expression between control vs. DIO mice with a tendency toward a decrease in the expression of enzymes involved in oxidative phosphorylation and fatty acid transport along with an increase in enzymes that positively influence glycolysis in DIO mice. Lastly, the antidiabetic drug metformin was found to inhibit OCR and alter the expression of several activation markers in CD4+ cells in vitro and in vivo, indicating that metformin may be an effective candidate to reverse T cell metabolic dysfunction and thereby restore T cell dysfunction in obesity. Research Support: National Institutes of Health grant R01-DK106090 and Derfner Foundation Research Award 2018.

An improved acridine orange staining of DNA/RNA

New tools are desirable to examine the metabolic state of individual cells within tissues. We proposed a fluorescence-based procedure consisting of acridine orange staining and fast green counterstaining (AO-FG) to improve the selectivity of the former for nucleic acids (acridine orange stains both DNA and RNA with different fluorescence colors), with no interference from proteins. We compared this test with the biochemical quantification of the relative amounts of RNA and DNA in selected rat ventral prostate samples and PC3 cells. The epithelium of the prostate gland is highly active metabolically for the production of secretions. Differences in AO-RNA staining were revealed and correlated with the metabolic state of the epithelium. Specificity was confirmed by RNase A. To assess how AO-FG staining correlates with the metabolic state of the cell, we cultured PC3 cells in different concentrations of glucose and measured the ratios between the amounts of RNA and DNA. In parallel, similar cultures were subjected to AO-FG, and the staining pattern correlated closely (r2=0.886) with the obtained biochemical results. The results confirmed that the combined use of AO and FG is useful for detecting DNA and RNA simultaneously, as well as for assessing quantitatively the transcriptional activity of individual cells and their changes in response to experimental manipulation.

Lactoferrin in Aseptic and Septic Inflammation.

Lactoferrin (Lf), a cationic glycoprotein able to chelate two ferric irons per molecule, is synthesized by exocrine glands and neutrophils. Since the first anti-microbial function attributed to Lf, several activities have been discovered, including the relevant anti-inflammatory one, especially associated to the down-regulation of pro-inflammatory cytokines, as IL-6. As high levels of IL-6 are involved in iron homeostasis disorders, Lf is emerging as a potent regulator of iron and inflammatory homeostasis. Here, the role of Lf against aseptic and septic inflammation has been reviewed. In particular, in the context of aseptic inflammation, as anemia of inflammation, preterm delivery, Alzheimer’s disease and type 2 diabetes, Lf administration reduces local and/or systemic inflammation. Moreover, Lf oral administration, by decreasing serum IL-6, reverts iron homeostasis disorders. Regarding septic inflammation occurring in Chlamydia trachomatis infection, cystic fibrosis and inflammatory bowel disease, Lf, besides the anti-inflammatory activity, exerts a significant activity against bacterial adhesion, invasion and colonization. Lastly, a critical analysis of literature in vitro data reporting contradictory results on the Lf role in inflammatory processes, ranging from pro- to anti-inflammatory activity, highlighted that they depend on cell models, cell metabolic status, stimulatory or infecting agents as well as on Lf iron saturation degree, integrity and purity.

Some reassembly required: Requirements for RAVE‐mediated reassembly of the yeast V‐ATPase

Lysosomes and vacuoles are the most acidic organelles in eukaryotic cells and play a central role in cellular metabolism – serving as a location that coordinates anabolic and catabolic processes. This coordination requires the activity of a highly conserved proton pump, the Vacuolar H+‐ATPase (V‐ATPase). Interestingly, both V‐ATPase activity and a cell's metabolic state are dependent on glucose levels. Glucose deprivation induces the V‐ATPase to disassemble into 3 distinct subcomplexes: V1, V1C, and Vo. V1 and V1C are released from Vo at the vacuolar membrane, and this disassembly silences both ATP hydrolysis in V1 and proton transport through Vo. Reassembly occurs rapidly but requires both glucose readdition and a conserved V‐ATPase‐specific assembly factor known as the RAVE (Regulator of the H+‐ATPase of Vacuolar and Endosomes) complex in yeast. The RAVE complex, composed of Rav1, Rav2, and Skp1, interacts with each V‐ATPase subcomplex and, upon glucose readdition, recruits cytosolic V1 and V1C to Vo at the vacuolar membrane to allow V‐ATPase reassembly (Smardon et al., (2015) J. Biol. Chem 290:27511). Although RAVE is essential for efficient V‐ATPase reassembly, how RAVE targets the vacuolar membrane in a glucose‐dependent manner and promotes V‐ATPase reassembly is not understood.Like the V1 subcomplex and V1C subunit, the RAVE complex is reversibly recruited to the vacuolar membrane in response to glucose. Interestingly, we found that RAVE requires the presence of Vo, but neither V1 nor V1C, for its glucose‐dependent vacuolar localization. We identified a 6‐amino acid, conserved motif within Rav1 that is essential for RAVE's vacuolar localization in vivo. In vitro, deletion of this motif diminishes binding between Rav1 and the cytosolic N‐terminal domain of Vo subunit Vph1. These data suggest that this motif is essential for RAVE to identify Vo subunit Vph1 at the vacuolar membrane, but they do not explain the release of RAVE from the membrane upon glucose deprivation. We seek to determine the signaling mechanism and structural changes involved in RAVE's glucose‐dependent activities. V‐ATPase activity, glycolytic enzymes, the Ras/cAMP pathway, PI(3,5)P2 levels, and cytoskeletal elements have all been implicated in V‐ATPase assembly. We are genetically or chemically silencing the activity of each of these factors and assessing the glucose‐dependent localization of GFP‐tagged RAVE subunits. Initial results indicate that RAVE cycles on and off the vacuolar membrane even in the presence of an assembled but inactive V‐ATPase mutant that is incapable of disassembly. Similar experiments will determine the effects of other factors. Detailed biochemical characterization of the RAVE complex has been thwarted by low expression levels of RAVE subunits. However, we can now express and purify milligram quantities of the RAVE complex alone or bound to V1. This will allow us to identify glucose‐sensitive interactions with RAVE in vitro and to test the hypothesis that RAVE undergoes a glucose‐sensitive conformation change that reversibly exposes the Rav1‐Vph1 binding site. This work addresses the molecular mechanisms governing RAVE‐mediated V‐ATPase reassembly and is essential to understanding the central role of the V‐ATPase in cellular metabolism.Support or Funding InformationNIH GM127364This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.