Quantitative analysis of mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) in mitochondria from mouse tissues and HeLa cells.

Mitochondrial calcium homeostasis plays critical roles in cell survival and aerobic metabolism in eukaryotes. The calcium uniporter is a highly selective calcium ion channel consisting of several subunits. Mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) are core subunits of the calcium uniporter required for calcium uptake activity in the mitochondria. Recent 3D structure analysis of the MCU-EMRE complex reconstituted in nanodiscs revealed that the human MCU exists as a tetramer forming a channel pore, with EMRE bound to each MCU at a 1 : 1 ratio. However, the stoichiometry of MCU and EMRE in the mitochondria has not yet been investigated. We here quantitatively examined the protein levels of MCU and EMRE in the mitochondria from mouse tissues by using characterized antibodies and standard proteins. Unexpectedly, the number of EMRE molecules was lower than that of MCU; moreover, the ratios between MCU and EMRE were significantly different among tissues. Statistical calculations based on our findings suggest that a MCU tetramer binding to 4 EMREs may exist, but at low levels in the mitochondrial inner membrane. In brain mitochondria, the majority of MCU tetramers bind to 2 EMREs; in mitochondria in liver, kidney, and heart, MCU tetramers bind to 1 EMRE; and in kidney and heart, almost half of MCU tetramers bound to no EMRE. We propose here a novel stoichiometric model of the MCU-EMRE complex in mitochondria.

mfapy: An open-source Python package for 13C-based metabolic flux analysis.

13C-based metabolic flux analysis (13C-MFA) is an essential tool for estimating intracellular metabolic flux levels in metabolic engineering and biology. In 13C-MFA, a metabolic flux distribution that explains the observed isotope labeling data was computationally estimated using a non-linear optimization method. Herein, we report the development of mfapy, an open-source Python package developed for more flexibility and extensibility for 13C-MFA. mfapy compels users to write a customized Python code by describing each step in the data analysis procedures of the isotope labeling experiments. The flexibility and extensibility provided by mfapy can support trial-and-error performance in the routine estimation of metabolic flux distributions, experimental design by computer simulations of 13C-MFA experiments, and development of new data analysis techniques for stable isotope labeling experiments. mfapy is available to the public from the Github repository (https://github.com/fumiomatsuda/mfapy).

Computational data mining method for isotopomer analysis in the quantitative assessment of metabolic reprogramming.

Measurement of metabolic flux levels using stable isotope labeling has been successfully used to investigate metabolic redirection and reprogramming in living cells or tissues. The metabolic flux ratio between two reactions can be estimated from the 13C-labeling patterns of a few metabolites combined with the knowledge of atom mapping in the complicated metabolic network. However, it remains unclear whether an observed change in the labeling pattern of the metabolites is sufficient evidence of a shift in flux ratio between two metabolic states. In this study, a data analysis method was developed for the quantitative assessment of metabolic reprogramming. The Metropolis-Hastings algorithm was used with an in silico metabolic model to generate a probability distribution of metabolic flux levels under a condition in which the 13C-labeling pattern was observed. Reanalysis of literature data demonstrated that the developed method enables analysis of metabolic redirection using whole 13C-labeling pattern data. Quantitative assessment by Cohen's effect size (d) enables a more detailed read-out of metabolic reprogramming information. The developed method will enable future applications of the metabolic isotopomer analysis to various targets, including cultured cells, whole tissues, and organs.

Sugar phosphate analysis with baseline separation and soft ionization by gas chromatography-negative chemical ionization-mass spectrometry improves flux estimation of bidirectional reactions in cancer cells.

Precise measurement of sugar phosphates in glycolysis and the pentose phosphate (PP) pathway for 13C-metabolic flux analysis (13C-MFA) is needed to understand cancer-specific metabolism. Although various analytical methods have been proposed, analysis of sugar phosphates is challenging because of the structural similarity of various isomers and low intracellular abundance. In this study, gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) is applied to sugar phosphate analysis with o-(2,3,4,5,6-pentafluorobenzyl) oxime (PFBO) and trimethylsilyl (TMS) derivatization. Optimization of the GC temperature gradient achieved baseline separation of sugar phosphates in 31 min. Mass spectra showed the predominant generation of fragment ions containing all carbon atoms in the sugar phosphate backbone. The limit of detection of pentose 5-phosphates and hexose 6-phosphates was 10 nM. The method was applied to 13C-labeling measurement of sugar phosphates for 13C-MFA of the MCF-7 human breast cancer cell line. 13C-labeling of sugar phosphates for 13C-MFA improved the estimation of the net flux and reversible flux of bidirectional reactions in glycolysis and the PP pathway.

Mass Spectrometry-Based Method to Study Inhibitor-Induced Metabolic Redirection in the Central Metabolism of Cancer Cells.

Cancer cells often respond to chemotherapeutic inhibitors by redirecting carbon flow in the central metabolism. To understand the metabolic redirections of inhibitor treatment on cancer cells, this study established a 13C-metabolic flux analysis (13C-MFA)-based method to evaluate metabolic redirection in MCF-7 breast cancer cells using mass spectrometry. A metabolic stationary state necessary for accurate 13C-MFA was confirmed during an 8-24 h window using low-dose treatments of various metabolic inhibitors. Further 13C-labeling experiments using [1-13C]glucose and [U-13C]glutamine, combined with gas chromatography-mass spectrometry (GC-MS) analysis of mass isotopomer distributions (MIDs), confirmed that an isotopic stationary state of intracellular metabolites was reached 24 h after treatment with paclitaxel (Taxol), an inhibitor of mitosis used for cancer treatment. Based on these metabolic and isotopic stationary states, metabolic flux distribution in the central metabolism of paclitaxel-treated MCF-7 cells was determined by 13C-MFA. Finally, estimations of the 95% confidence intervals showed that tricarboxylic acid cycle metabolic flux increased after paclitaxel treatment. Conversely, anaerobic glycolysis metabolic flux decreased, revealing metabolic redirections by paclitaxel inhibition. The gap between total regeneration and consumption of ATP in paclitaxel-treated cells was also found to be 1.2 times greater than controls, suggesting ATP demand was increased by paclitaxel treatment, likely due to increased microtubule polymerization. These data confirm that 13C-MFA can be used to investigate inhibitor-induced metabolic redirection in cancer cells. This will contribute to future pharmaceutical developments and understanding variable patient response to treatment.

Investigation of useful carbon tracers for 13C-metabolic flux analysis of Escherichia coli by considering five experimentally determined flux distributions.

The 13C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13C-labeled glucose for 13C-metabolic flux analysis (13C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli, the best fitted flux distribution and the 95% confidence interval were estimated by the 13C-MFA procedure. A comparison of the precision scores showed that [1, 2-13C]glucose and a mixture of [1-13C] and [U-13C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1-13C], and [U-13C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13C-MFA experiments.

Photoinduced electron transfer promoted radical ring expansion and cyclization reactions of α-(ω-carboxyalkyl) ß-keto esters.

Photoinduced electron transfer (PET) promoted decarboxylation of α-(ω-carboxyalkyl) ß-keto esters undergoes radical ring expansion and cyclization reactions. This mild and environmentally friendly method can provide one-carbon expanded γ-keto esters and bicyclic alcohols, and the product distribution is strongly dependent on the length of the alkyl chain containing the terminal carboxylate group.

Radical photocyclization route for macrocyclic lactone ring expansion and conversion to macrocyclic lactams and ketones.

A new method for the synthesis of macrocyclic lactones, lactams, and ketones, which utilizes photoinduced intramolecular radical cyclization reactions of substrates containing tethered carboxylic acids and α,ß-unsaturated carbonyl moieties, has been uncovered. Photocyclization of the carboxylic acids tethered acrylate ester, which were prepared starting from the macrocyclic lactones, gave the two-carbon elongated macrocyclic lactones via decarboxylation. Similar photoreactions of carboxylic acid tethered acryl amide or α,ß-unsaturated ketone moieties, which were also prepared starting from the macrocyclic lactones, produced macrocyclic lactams or ketones, respectively. The simple approach can be readily applied to the preparation of a variety of macrocyclic lactones, lactams, and ketones with tunable ring sizes.

Mathematical modelling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells II.

The Drosophila posterior midgut epithelium mainly consists of intestinal stem cells (ISCs); semi-differentiated cells, i.e. enteroblasts (EBs); and two types of fully differentiated cells, i.e. enteroendocrine cells (EEs) and enterocytes (ECs), which are controlled by signalling pathways. In [M. Kuwamura, K. Maeda, and T. Adachi-Yamada, Mathematical modeling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells I, J. Biol. Dyn. 4 (2009), pp. 248-257], on the basis of the functions of the Wnt and Notch signalling pathways, we studied the regulatory mechanism for the proliferation and differentiation of ISCs under the assumption that the Wnt proteins are supplied from outside the cellular system of ISCs. In this paper, we experimentally show that the Wnt proteins are specifically expressed in ISCs, EBs, and EEs, and theoretically show that the cellular system of ISCs can be self-maintained under the assumption that the Wnt proteins are produced in the cellular system of ISCs. These results provide a useful basis for determining whether an environmental niche is required for maintaining the cellular system of tissue stem cells.

Influence of solvent, electron acceptors and arenes on photochemical decarboxylation of free carboxylic acids via single electron transfer (SET).

Single electron transfer (SET)-photochemical decarboxylation of free carboxylic acids was performed in a polar solvent using several arenes such as phenanthrene, naphthalene, 1-methylnaphthalene, biphenyl, triphenylene, and chrysene in the presence of various electron acceptors such as 1,2-, 1,3-, and 1,4-dicyanobenzenes, methyl 4-cyanobenzoate, and 1,4-dicyanonaphthalene. The decarboxylation reaction was influenced by the arenes, electron acceptors, and solvent. The best result was achieved by the photoreaction using biphenyl and 1,4-dicyanonaphthalene in aqueous acetonitrile.

Mathematical modelling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells I.

We study the proliferation and differentiation of stem cells in the Drosophila posterior midgut epithelium, which mainly consists of intestinal stem cells (ISCs); semi-differentiated cells, i.e. enteroblasts (EBs); and two types of fully differentiated cells, i.e. enteroendocrine cells (EEs) and enterocytes (ECs). The cellular system of ISCs is controlled by Wnt and Notch signalling pathways. In this article, we experimentally show that EBs are not capable of efficiently differentiating into ECs in the absence of Wnt signalling. On the basis of the experimental results and known facts, we propose a scheme and a simple ordinary differential equation (ODE) model for the proliferation and differentiation of ISCs. This is a first step towards understanding the universal mechanism for the maintenance of the cellular system of tissue stem cells controlled by signalling pathways.

RNAi-mediated knockdown showing impaired cell survival in Drosophila wing imaginal disc.

The genetically amenable organism Drosophila melanogaster has been estimated to have 14,076 protein coding genes in the genome, according to the flybase release note R5.13 (http://flybase.bio.indiana.edu/static_pages/docs/release_notes.html). Recent application of RNA interference (RNAi) to the study of developmental biology in Drosophila has enabled us to carry out a systematic investigation of genes affecting various specific phenotypes. In order to search for genes supporting cell survival, we conducted an immunohistochemical examination in which the RNAi of 2,497 genes was independently induced within the dorsal compartment of the wing imaginal disc. Under these conditions, the activities of a stress-activated protein kinase JNK (c-Jun N-terminal kinase) and apoptosis-executing factor Caspase-3 were monitored. Approximately half of the genes displayed a strong JNK or Caspase-3 activation when their RNAi was induced. Most of the JNK activation accompanied Caspase-3 activation, while the opposite did not hold true. Interestingly, the area activating Caspase-3 was more broadly seen than that activating JNK, suggesting that JNK is crucial for induction of non-autonomous apoptosis in many cases. Furthermore, the RNAi of essential factors commonly regulating transcription and translation showed a severe and cell-autonomous apoptosis but also elicited another apoptosis at an adjacent area in a non-autonomous way. We also found that the frequency of apoptosis varies depending on the tissues.

Inter- and intramolecular addition reactions of electron-deficient alkenes with alkyl radicals, generated by SET-photochemical decarboxylation of carboxylic acids, serve as a mild and efficient method for the preparation of gamma-amino acids and macrocyclic lactones.

Inter- and intramolecular additions of alkyl radicals, generated by SET photochemical decarboxylation reactions of free carboxylic acids, to electron-deficient alkenes take place under mild conditions as part of efficient routes for the formation of N-Boc gamma-amino acids and macrocyclic lactones.

E-cadherin prolongs the moment for interaction between intestinal stem cell and its progenitor cell to ensure Notch signaling in adult Drosophila midgut.

Intestinal stem cells (ISCs) are required for maintenance of the proper cell composition in the adult intestine. To ensure permanent recruitment of newly differentiated cells, the ISC undergoes asymmetric cell division that generates an ISC itself and a progenitor cell. In the Drosophila midgut, cell fate for the absorptive cell is determined by Notch (N) signal in the progenitor cells that receive a ligand Delta (Dl) produced by the ISCs. Although most of the ISCs and progenitor cells are distantly located, they should retain their attachment when N is activated because the Dl-N interaction requires cell adhesion. Furthermore, N cannot be activated before completion of cell division. Thus, the moment after cell division and before cell separation should be prolonged for certain N activation, although the mechanism for this remains unclear. Here, we demonstrate that E-cadherin (E-cad) is required for stable attachment between the two cells. When E-cad does not function, N is not activated and cell differentiation is attenuated. We also show that the ISC tumor by N inactivation is assisted by a defect in E-cad down-regulation. These findings reveal one of the normal N functions used to inhibit tumorigenesis through lowering of E-cad for proper midgut cell turnover.

Drosophila T-box transcription factor Optomotor-blind prevents pathological folding and local overgrowth in wing epithelium through confining Hh signal.

Aberration of morphogen signaling leads directly to inappropriate cell differentiation and secondarily causes various pathological phenotypes such as abnormal morphogenesis and tumorigenesis. However, mechanisms for linking morphogen signaling and the higher order phenotypes have not been fully elucidated. Here we focus on the Drosophila T-box gene optomotor-blind (omb), a transcriptional target of a long-range morphogen Decapentaplegic (Dpp). Genetic analyses of omb function revealed that a negative feedback loop, where omb plays a crucial role, exists between Dpp and its upstream regulator Hedgehog (Hh), a short-range morphogen. Consequently, dysfunction of omb elicits hyperactivation of Hh signaling that causes an ectopic folding and local overgrowth in the wing columnar epithelium, neither of which are the direct results of reduced Dpp response. In the case of the local overgrowth, it was never seen in mutants for thick veins (tkv) encoding a Dpp receptor, suggesting that the Dpp signaling pathway is divided into two antagonistic branches, one of which contains Omb. Thus defect in feedback between the two morphogens explains both phenotypes, and disruption of a balance between the morphogen targets further accounts for the local overgrowth. These are the mechanisms for generating secondary phenotypes when a single signaling factor Omb fails to function.