Habits and Binds of Mathematics Education in the Anthropocen

<p>(a) A signaling network for cell death regulation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165049#pone.0165049.ref011" target="_blank">11</a>]. Cell death is enhanced due to DNA damage while inhibited by the signals transmitted from EGFR. The arrow shape represents activation while a flat-head edge means inhibition. Pink nodes denote the species that have experimental measurements. (b) The simulation results of DNA Damage and Cell Death.</p

Supplemental tables from Rhythmicity and waves in the cortex of single cells

Supplemental Table 1: Summary of the properties of periodic travelling waves in the single cells; Supplemental Table 2: Summary of actin wave velocity (unit: μm/sec)

Rapid Analysis of Glycolytic and Oxidative Substrate Flux of Cancer Cells in a Microplate

<div><p>Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents. In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively. These analyses led to three interesting observations: 1) both cell lines respired effectively with substantial endogenous substrate respiration; 2) SF188f cells underwent a significant shift from glycolytic to oxidative metabolism, along with a high rate of glutamine oxidation relative to SF188s cells; and 3) the mitochondrial proton leak-linked respiration of SF188f cells increased significantly compared to SF188s cells. It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation. Taken together, these rapid, sensitive and high-throughput substrate flux analysis methods introduce highly valuable approaches for developing a greater understanding of genetic and epigenetic pathways that regulate cellular metabolism, and the development of therapies that target cancer metabolism.</p></div

Lowered basal glycolytic flux but acquired glycolytic capacity in SF188f cells compared with SF188s cells.

<p>A. ECAR response of SF188s and SF188f cells to glucose (10 mM), oligomycin (1 µM), and 2-DG (100 mM). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. ECAR values were normalized to mpH/10⁴ cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux and glycolytic capacity of SF188s and SF188f cells normalized to mpH/min/10,000 cells. * p<0.05.</p

Assaying glutamine oxidation and demonstrating transaminase pathway activity.

<p>A. Schematic illustration of biochemical pathway for glutamine oxidation in the mitochondria. B. Kinetic OCR response in SF188f cells to glutamine (4 mM). SF188f cells were plated at 20,000 cells/well in XF24 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. The OCR value was not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. C. OCR response (% of baseline) in SF188f cells to glutamine (4 mM) and AOA (100 µM). Glutamine-induced OCR reached 60% over the baseline (OCR at measurement 6 divided by that at measurement 3) while AOA addition reduced it to 20% (OCR at Measurement 9 divided by measurement 3). SF188f cells were plated at 20,000 cells/well in XF24 V7 cell culture plates 24–28 hours before the assays. The % OCR was plotted using measurement 3 as the baseline. The assay medium was the substrate-free base medium. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.</p

Analyzing Glycolytic flux.

<p>A. Schematic illustration of the glycolytic pathway. NADH produced in the cytosol as glucose is converted to pyruvate and is regenerated by LDH in the cytosol. B. Kinetic ECAR response of SF188s cells to glucose (10 mM) and 2-DG (100 mM) or oxamate (100 mM), respectively. SF188s cells were plated at 30,000 cells/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was substrate-free base medium (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109916#s2" target="_blank">Material and Methods</a>) supplemented with 2 mM glutamine. The ECAR value was not normalized. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 4. C. ECAR response of HeLa cells to glucose (10 mM), 2-DG (100 mM) and antimycin (1 µM). Insert: the OCR response in the same experiments showing the Crabtree effect and that glucose did not increase OCR. HeLa cells were plated at 30,000/well in XF24 cell culture plates 24–28 hours prior to the assays. ECAR or OCR values were not normalized. The assay medium was substrate-free base medium (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109916#s2" target="_blank">Material and Methods</a>) supplemented with 2 mM glutamine. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 5.</p

Enhanced glutamine oxidation and activation of fatty acid oxidation in SF188f cells.

<p>A. Kinetic OCR response of SF188s and SF188f cells to glutamine (4 mM) (left panel). Insert: calculated glutamine oxidation rate of SF188s and SF188f cells. OCR response (% of baseline) to glutamine and AOA (100 µM) (right panel). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. The OCR values were normalized to pmoles/min/10⁴ cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B and C. OCR response of SF188s and SF188f cells to palmitate-BSA (150 mM) (B) and octanoate (1 mM) (C). SF188s and SF188f cells were plated at 20,000 and 15,000 cells/well, respectively, in XF96 V3 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. Fatty acid oxidation was expressed as % OCR plotted using measurement 3 as the baseline. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 6.</p

Determining fatty acid oxidation.

<p>A. Schematic illustration of biochemical pathway of fatty acid oxidation. B. Kinetic OCR response of SF188f cells to palmitate (150 µM). C. Kinetic OCR response in SF188f cells to octanoate (1 mM). SF188f cells were plated at 15,000 cells/well in XF96 V3 cell culture plates 24–28 hours prior to the assays. Assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. The OCR value was not normalized. A representative experiment out of four is shown here. Each data point represents mean ± SD, n = 6.</p

Markedly reprogrammed mitochondrial bioenergetic machinery of SF188s and SF188f cells.

<p>A. Kinetic OCR response of SF188s and SF188f cells to oligomycin (1 and 2 µM respectively) to determine ATP coupled respiration, FCCP (0.3 µM) to establish maximal respiratory capacity, and myxothiazol (1 µM) and rotenone (1 µM) cocktail) to define mitochondrial respiration (left). Calculated ATP-coupled respiration, proton leak-linked respiration, maximal mitochondrial respiratory capacity (right). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 25 mM glucose, 6 mM glutamine and 1 mM pyruvate. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. OCR values were normalized to pmoles/10⁴ cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 3. *p<0.05. B. Kinetic OCR response (% of baseline, baseline = measurement 3) of SF188f (right) and SF188s cell (left) with distribution of ATP-coupled respiration (% of oligomycin-sensitive at measurement 4) and proton leak-linked respiration (oligomycin-resistant at measurement 4 but myxothiazol-sensitive mitochondrial OCR at measurement 12) and non-mitochondria respiration (myxothiazol-resistant OCR at measurement 12).</p

Schematic illustration of cellular metabolism pathways along with assays of glucose, glutamine, and fatty acid oxidation, glycolytic flux and mitochondrial bioenergetics.

<p>Schematic illustration of cellular metabolism pathways along with assays of glucose, glutamine, and fatty acid oxidation, glycolytic flux and mitochondrial bioenergetics.</p