Nicotinamide Adenine Dinucleotide Hydride Research Articles

On the coupling of intracellular to glycolytic oscillations in yeast.

We have investigated the interplay between glycolytic oscillations and intracellular concentration in the yeastSaccharomyces cerevisiae. Intracellular concentration was measured using the fluorophore potassium-binding benzofuranisophthalate(PBFI). We found that is an essential ion for the occurrence of glycolytic oscillations and that intracellular concentration oscillates synchronously with other variables such as nicotinamide adenine dinucleotide hydride (NADH), intracellular adenosine triphosphate (ATP), and mitochondrial membrane potential. We also investigated if glycolysis and intracellular concentration oscillate in a number of yeast strains with mutations in transporters in the plasma membrane, mitochondrial membrane and in the vacuolar membrane. Most of these strains are still capable of showing glycolytic oscillations, but two strains are not: (i) a strain with a deletion in the mitochondrial Mdm38p transporter and (ii) a strain with deletion of the late endosomal Nhx1p ( ) transporter. In these two mutant strains intracellular concentration seems to be low, indicating that the two transporters may be involved in transport of into the cytosol. In the strain, Mdm38p oscillations in glycolysis could be restored by addition of the exchange ionophore nigericin. Furthermore, in two nonoscillating mutant strains with a defective V-ATPase and deletion of the Arp1p protein the intracellular is relatively high, suggesting that the V-ATPase is essential for transport of out of the cytosol and that the cytoskeleton may be involved in binding to reduce the concentration of free ion in the cytosol. Analyses of the time series of oscillations of NADH, ATP, mitochondrial membrane potential, and potassium concentration using data-driven modeling corroborate the conjecture that ion is essential for the emergence of oscillations and support the experimental findings using mutant strains.

A vendor-neutral EPI sequence for hyperpolarized 13C MRI.

To develop a flexible, vendor-neutral EPI sequence for hyperpolarized 13C metabolic imaging. An open-source EPI sequence consisting of a metabolite-specific spectral-spatial RF excitation pulse and a customizable EPI readout was created using the Pulseq framework. To explore the flexibility of our sequence, we tested several versions of the sequence including a symmetric 3D readout with different spatial resolutions for each metabolite (1.0 cm3 and 1.5 cm3). A multichamber phantom constructed with a Shepp-Logan geometry, containing two chambers filled with either natural abundance 13C compounds or hyperpolarized (HP) [1-13C]pyruvate, was used to test each sequence. For experiments involving HP [1-13C]pyruvate, a single chamber was prefilled with nicotinamide adenine dinucleotide hydride and lactate dehydrogenase to facilitate the conversion of [1-13C]pyruvate to [1-13C]lactate. All experiments were performed on a Siemens Prisma 3T scanner. All the sequence variations localized natural-abundance 13C ethylene glycol and methanol to the appropriate compartment of the multichamber phantom. [1-13C]pyruvate was detectable in both chambers following the injection of HP [1-13C]pyruvate, whereas [1-13C]lactate was only found in the chamber containing nicotinamide adenine dinucleotide hydride and lactate dehydrogenase. The conversion rate from [1-13C]pyruvate to [1-13C]lactate (kPL) was 0.01 s-1 (95% confidence interval [0.00, 0.02]). We have developed and tested a vendor-neutral EPI sequence for imaging HP 13C agents. We have made all of our sequence creation and image reconstruction code freely available online for other investigators to use.

New Tiaoxin Recipe Alleviates Energy Metabolism Disorders in an APPswe/PS1DE9 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a typical neurodegenerative disease with a complex etiology. Until now, there has been no effective treatment available for AD; however, improving energy dysmetabolism, the key pathological event in the early stage of AD, can effectively delay the progression of AD. This paper aims to investigate the therapeutic effect and potential mechanism of the new Tiaoxin recipe on early AD. APP/PS1 mice were divided into a model group, a new Tiaoxin recipe group, and a donepezil group, and C57/BL mice were used for the control group. Mouse cognitive and learning abilities were tested using the Morris water maze test and a new object-recognition experiment. The 42 amino acid form of amyloid β peptide (Aβ1-42) content was detected by enzyme-linked immunosorbent assay, the senile plaque area was detected by thioflavin S staining, and the senescence- associated β-galactosidase (SA-β-gal)-positive area was detected by chemical staining. Also, the adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), and nicotinamide adenine dinucleotide hydride (NADH) contents were detected using a biochemical method, and the cluster of differentiation 38 (CD38) and silent mating-type information regulation 2 homolog 3 (SIRT3) protein expression levels were detected by immunofluorescence and Western blot analysis. Compared with those of the control group, the learning and memory abilities of the model group were impaired; the senile plaque deposition, Aβ1-42 content, and SA-βgal-positive staining area were increased; the ATP concentration, NAD+ concentration, and NAD+/NADH ratio were decreased; the CD38 protein expression level was increased; and the SIRT3 protein expression level was decreased. Following intervention with the new Tiaoxin recipe, the learning and memory abilities were improved; the senile plaque deposition, Aβ1-42 content, and SA-βgal-positive area were reduced; the ATP concentration, NAD+ concentration, and NAD+/NADH ratio were increased; CD38 protein expression was decreased, and SIRT3 protein expression was increased. This study shows that the new Tiaoxin Recipe can improve cognitive ability and reduce the Aβ1-42 content and senile plaque deposition in APP/PS1 mice, which may occur through the downregulation of CD38 protein expression, upregulation of SIRT3 protein expression, restoration of the NAD+ level, promotion of ATP synthesis, mitigation of energy metabolism disorders.

A Mechanism Underpinning the Bioenergetic Metabolism-Regulating Function of Gold Nanocatalysts.

Bioenergetic deficits are known to be significant contributors to neurodegenerative diseases. Nevertheless, identifying safe and effective means to address intracellular bioenergetic deficits remains a significant challenge. This work provides mechanistic insights into the energy metabolism-regulating function of colloidal Au nanocrystals, referred to as CNM-Au8, that are synthesized electrochemically in the absence of surface-capping organic ligands. When neurons are subjected to excitotoxic stressors or toxic peptides, treatment of neurons with CNM-Au8 results in dose-dependent neuronal survival and neurite network preservation across multiple neuronal subtypes. CNM-Au8 efficiently catalyzes the conversion of an energetic cofactor, nicotinamide adenine dinucleotide hydride (NADH), into its oxidized counterpart (NAD+ ), which promotes bioenergy production by regulating the intracellular level of adenosine triphosphate. Detailed kinetic measurements reveal that CNM-Au8-catalyzed NADH oxidation obeys Michaelis-Menten kinetics and exhibits pH-dependent kinetic profiles. Photoexcited charge carriers and photothermal effect, which result from optical excitations and decay of the plasmonic electron oscillations or the interband electronic transitions in CNM-Au8, are further harnessed as unique leverages to modulate reaction kinetics. As exemplified by this work, Au nanocrystals with deliberately tailored structures and surfactant-free clean surfaces hold great promise for developing next-generation therapeutic agents for neurodegenerative diseases.

Pelargonium sidoides extract mediates nephrotoxicity through mitochondrial malfunction and cytoskeleton destabilization.

We investigated the cytotoxic effect of Pelargonium sidoides extract on Madin-Darby canine kidney (MDCK) cells. P. sidoides extract decreased the cell viability in a dose dependent manner (> 0.2%). The extract of P. sidoides decreased the mitochondrial action potential, increased the number of reactive oxygen species (ROS) inside the cell, and caused nicotinamide adenine dinucleotide hydride (NADH) to be released, all of which are signs of mitochondrial dysfunction. The results of unbiased mRNA sequencing showed that 0.3% P. sidoides extract upregulates the apoptosis-related gene (BBC3). This finding was supported by immunoblot analysis of apoptosis signal pathways, which included Bcl-2, Bax, cytochrome C (CytC), cleaved caspase 3 (CC3), cleaved caspase 7 (CC7), cleaved caspase 9(CC9) and cleaved PARP (CP). It is interesting to note that the elevated levels of Bax, CytC, CC3, CC7, and CC9, as well as CP, were suppressed by N-acetyl-L-cysteine (NAC) pretreatment, which points to ROS-mediated apoptosis. The small GTPases, RhoA, and Rac1/cdc42-GTP-bound active form were all lowered when P. sidoides extract was used. Also, RhoA-related cytoskeleton signals (ROCK, p-LIMK1/2, p-cofilin) and Rac1/cdc42-related signals (N-WASP, WAVE-2) were inhibited by P. sidoides extract. NAC or RhoA/Rac1/cdc42 activator pretreatment reduced P. sidoides extract-induced actin destabilization. In this work, P. sidoides extract promotes apoptosis by causing mitochondrial dysfunction and cytoskeleton disassembly.

Morphological and molecular phylogenetic data reveal another new species of bent-toed gecko (Cyrtodactylus Gray: Squamata: Gekkonidae) from Mizoram, India

ABSTRACT A new species of Cyrtodactylus is described herein on the basis of morphological and phylogenetic evidence inferred from the mitochondrial nicotinamide adenine dinucleotide hydride dehydrogenase subunit 2 gene from Mizoram, Northeast India. The new species is distinguished from its congeners by an adult snout-vent length of 62.6–68.6 mm; 10–11 supralabials; 9–11 infralabials; dorsal tubercles rounded, conical to weakly keeled and in 19–20 longitudinal rows; 32–36 paravertebral tubercles between the level of axilla and groin; 32–39 mid-ventral scale rows across the ventral region; 6 precloacal pores in males and 0–6 precloacal pits in females; 14–18 subdigital lamellae under toe IV; no single row of transversely enlarged subcaudal scales; indistinct pattern of alternating dark brown blotches on the dorsum of the body. Morphologically and genetically, the new species is recovered as the sister species to C. aaronbaueri, but is genetically distinct by an uncorrected p-distance of 0.099–0.1, and morphologically in the number of precloacal pores, mid-ventral scale rows, paravertebral tubercles on the trunk, dorsal tubercle rows, dorsal spot, and subdigital lamellae on pes. http://www.zoobank.org/urn:lsid:zoobank.org:pub:8E9144FA-EFAF-4C7D-B64F-667706643EC5

Light-Harvesting Artificial Cells Containing Cyanobacteria for CO2 Fixation and Further Metabolism Mimicking.

The bottom-up constructed artificial cells help to understand the cell working mechanism and provide the evolution clues for organisms. The energy supply and metabolism mimicry are the key issues in the field of artificial cells. Herein, an artificial cell containing cyanobacteria capable of light harvesting and carbon dioxide fixation is demonstrated to produce glucose molecules by converting light energy into chemical energy. Two downstream "metabolic" pathways starting from glucose molecules are investigated. One involves enzyme cascade reaction to produce H2 O2 (assisted by glucose oxidase) first, followed by converting Amplex red to resorufin (assisted by horseradish peroxidase). The other pathway is more biologically relevant. Glucose molecules are dehydrogenated to transfer hydrogens to nicotinamide adenine dinucleotide (NAD+ ) for the production of nicotinamide adenine dinucleotide hydride (NADH) molecules in the presence of glucose dehydrogenase. Further, NADH molecules are oxidized into NAD+ by pyruvate catalyzed by lactate dehydrogenase, meanwhile, lactate is obtained. Therefore, the cascade cycling of NADH/NAD+ is built. The artificial cells built here pave the way for investigating more complicated energy-supplied metabolism inside artificial cells.

P-026 Lack of association between single polymorphic variants of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase 3, and 4L (MT-ND3 and MT-ND4L) and male infertility

Abstract Study question Is there any association between male infertility and the polymorphic variants of Mitochondrial Nicotinamide Adenine Dinucleotide Hydride dehydrogenase (NADH) Subunit 3 and 4L (MT-ND3 and MT-ND4L)? Summary answer The genotypes and allele frequencies of the study population have shown a lack of statistically significant association between MT-ND3 and MT-ND4L SNPs and male infertility. What is known already Male infertility is a multifactorial condition associated with different genetic abnormalities in at least 15%–30% of cases. The rate of mutations in the mtDNA is high due to the lack of histones and DNA repair mechanisms. Moreover, mtDNA is more vulnerable compared to nuclear DNA due to the lack of an efficient repairing system and high exposure to oxidative species produced by the mitochondria. Eventually, this leads to abnormal sperm function, structure and even infertility. Study design, size, duration Prospective study was carried out between 2018 and 2019. 112 semen samples were collected in this study. Participants/materials, setting, methods The present study was carried out at the department of Obstetrics and Gynecology, University of Saarland,Germany. After semen analysis, samples were purified. Mitochondrial DNA was extracted using a QIAamp DNA Mini Kit, then, the mitochondrial DNA was amplified by using the REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify MT-ND3 and MT-ND4L genes. After that samples were purified and sequenced using the Sanger method in the Microsynth Seq Lab in Germany. Main results and the role of chance Eight single nucleotide polymorphisms (SNPs) in MT-ND3 (rs2853826, rs28435660, rs193302927, rs28358278, rs41467651, rs3899188, rs28358277 and rs28673954) and seven SNPs in MT-ND4L (rs28358280, rs28358281, rs28358279, rs2853487, rs2853488, rs193302933 and rs28532881) were detected and genotyped. The genotypes and allele frequencies of the study population have shown a lack of statistically significant association between MT-ND3 and MT-ND4L SNPs and male infertility. Besides, no statistically significant difference was found between the asthenozoospermia, oligozoospermia, teratozoospermia, asthenoteratozoospermia, oligoasthenoteratozoospermia and oligoteratozoospermia subgroups of subfertile males. However, rs28358278 genotype of the MT-ND3 gene was reported in the subfertile group but not in the fertile group, which implies a possible role of this SNP in male infertility. Limitations, reasons for caution The size number of the study samples. Wider implications of the findings Further studies are required to evaluate these findings. Moreover, the subfertile individuals who exhibit a polymorphism at rs28358278 require further monitoring and evaluation. Trial registration number Basic Science

Thyroid Cancer-Associated Mitochondrial DNA Mutation G3842A Promotes Tumorigenicity via ROS-Mediated ERK1/2 Activation.

Mitochondrial DNA (mtDNA) mutations have been identified in various human cancers, including thyroid cancer. However, the relationship between mtDNA and thyroid cancer remains unclear. Previous studies by others and us strongly suggested that mtDNA mutations in complex I may participate in thyroid cancer processes according to sequencing results of thyroid cancer tissue, although the associated pathogenic processes remain unknown. Here, to investigate whether mtDNA mutations contribute to thyroid cancer, we reanalyzed our sequencing results and characterized thyroid cancer-associated mutations in the mitochondrial complex. The results identified the highest mutation frequencies in nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase subunit 4 gene (ND4) and cytochrome c oxidase subunit 1 gene (COI), which also harbored the highest rates of G > A substitutions, with most of the mutations resulting in changes in the polarity of amino acids. We then established cybrids containing the G3842A mutation identified in papillary thyroid carcinoma, which revealed it as a mutation in NADH dehydrogenase subunit 1 gene (ND1) and is previously reported in follicular thyroid carcinoma, thereby suggesting a possibly pathogenic role in thyroid carcinoma. Additionally, we found that the G3842A mutation accelerates tumorigenicity and decreases the abundance and activity of mitochondrial complex I, the oxygen consumption rate, and adenosine triphosphate levels. By contrast, the levels of reactive oxygen species (ROS) were increased to activate extracellular signal-regulated kinase (ERK1/2) signaling, which contributed to tumorigenicity. These findings suggest for the first time that mtDNA mutations help drive tumor development and that G3842A may represent a new risk factor for thyroid cancer. Furthermore, our findings indicate that drugs targeting ROS and ERK1/2 may serve as a viable therapeutic strategy for thyroid cancer.

A Novel Approach to Develop Lager Yeast with Higher NADH Availability to Improve the Flavor Stability of Industrial Beer.

Flavor stability is important for beer quality and extensive efforts have been undertaken to improve this. In our previous work, we proved a concept whereby metabolic engineering lager yeast with increased cellular nicotinamide adenine dinucleotide hydride (NADH) availability could enhance the flavor stability of beer. However, the method for breeding non-genetically modified strains with higher NADH levels remains unsolved. In the current study, we reported a novel approach to develop such strains based on atmospheric and room temperature plasma (ARTP) mutagenesis coupled with 2,4-dinitrophenol (DNP) selection. As a result, we obtained a serial of strains with higher NADH levels as well as improved flavor stability. For screening an optimal strain with industrial application potential, we examined the other fermentation characteristics of the mutants and ultimately obtained the optimal strain, YDR-63. The overall fermentation performance of the strain YDR-63 in pilot-scale fermentation was similar to that of the parental strain YJ-002, but the acetaldehyde production was decreased by 53.7% and the resistance staling value of beer was improved by 99.8%. The forced beer aging assay further demonstrated that the favor stability was indeed improved as the contents of 5-hydroxymethylfurfural in YDR-63 was less than that in YJ-002 and the sensory notes of staling was weaker in YDR-63. We also employed this novel approach to another industrial strain, M14, and succeeded in improving its flavor stability. All the findings demonstrated the efficiency and versatility of this new approach in developing strains with improved flavor stability for the beer industry.

P–542 Relationship between the single nucleotides polymorphisms in Mitochondrial Nicotinamide Adenine Dinucleotide Hydride dehydrogenase (NADH) Subunit 4 gene (MT-ND4) and male infertility

Abstract Study question Is there any association between male infertility and the polymorphic variants of Mitochondrial Nicotinamide Adenine Dinucleotide Hydride dehydrogenase (NADH) Subunit 4 (MT-ND4)? Summary answer Our findings suggested that male infertility was correlated to rs2853495 and rs869096886 SNPs in MTND4. What is known already The rate of mutations in the mtDNA, the powerhouse of the cell, is high due to the lack of histones and DNA repair mechanisms. Therefore, mutations that occur in the mitochondrial genome, play a major role in some human genetic disorders. 15 - 30% of male infertility are related to genetic predisposition. Sperm containing defective mitochondria cannot effectively produce ATP and more likely to produce reactive oxygen species (ROS) and free radicals, thereby causing a defect in mtDNA, make trouble energy, and deteriorate motility and fertility. Study design, size, duration: A prospective study carried out between 2018 and 2019. 112 semen samples were collected in this study. Participants/materials, setting, methods The present study was carried out at the department of Obstetrics and Gynecology, University of Saarland, Germany. Samples were divided into 68 subfertile and 44 fertile men. Mitochondrial DNA was extracted using a QIAamp DNA Mini Kit, after that the mtDNA was amplified by using REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify MT-ND4 gene. Then, samples were purified and sequenced using the Sanger method in the Microsynth Seq lab, Germany. Main results and the role of chance The genotypes frequencies of the study population showed a statistically significant association between rs2853495 G>A (Gly320Gly) and male infertility (P = 0.0351). Similarly, the allele frequency test showed that rs2853495 G>A (Gly320Gly) and rs869096886 A>G (Leu164Leu) were significantly associated with male infertility (adjusted OR = 2.616, 95% CI = 1.374 - 4.983, P = 0.0028; adjusted OR = 2.237, 95% CI = 1.245 - 4.017, P = 0.0073, respectively). On the other hand, no statistically significant difference was observed between the asthenozoospermia, oligozoospermia, teratozoospermia, asthenoteratozoospermia, oligoasthenoteratozoospermia, oligoteratozoospermia subgroups of subfertile males and the fertile ones. Limitations, reasons for caution The size number of the study samples. Wider implications of the findings: A larger prospective study will be required to confirm these associations of mitochondrial gene polymorphisms rs2853495 and rs869096886 in MT-ND4 and male infertility and to clarify the definite effect of the mitochondrial genetic variations on male infertility. Trial registration number Not applicable

Mitochondrial nicotinamide adenine dinucleotide hydride dehydrogenase (NADH) subunit 4 (MTND4) polymorphisms and their association with male infertility

PurposeThe purpose of the present study was to determine the relationship between infertility and the polymorphisms of mitochondrial NADH dehydrogenase subunit 4 (MTND4) by spermatozoa analysis in fertile and subfertile men.MethodsSamples were divided into 68 subfertile men (case group) and 44 fertile men (control group). After semen analysis, samples were purified. The whole genome was extracted using a QIAamp DNA Mini Kit and the mitochondrial DNA was amplified by using the REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify the MT-ND4 gene. Then, samples were purified and sequenced using the Sanger method.ResultsTwenty-five single-nucleotide polymorphisms (SNPs) were identified in the MTND4 gene. The genotype frequencies of the study population showed a statistically significant association between rs2853495 G>A (Gly320Gly) and male infertility (P = 0.0351). Similarly, the allele frequency test showed that rs2853495 G>A (Gly320Gly) and rs869096886 A>G (Leu164Leu) were significantly associated with male infertility (adjusted OR = 2.616, 95% CI = 1.374–4.983, P = 0.002; adjusted OR = 2.237, 95% CI = 1.245–4.017, P = 0.007, respectively).ConclusionIn conclusion, our findings suggested that male infertility was correlated with rs2853495 and rs869096886 SNPs in MTND4.

Green tea extract increases the quality and reduced DNA mutation of post-thawed Kacang buck sperm

The study aimed to determine the addition of green tea extract (GTE) in extender on the quality and DNA mutation of post-thawed Kacang buck sperm. The sperm DNA mutation was observed on nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase 1 (ND1) of mitochondrial Deoxyribonucleic Acid (mtDNA). A pool of 12 Kacang buck ejaculates was diluted in skim milk-egg yolk extender contained 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 group, respectively. Each of the aliquot groups was packaged in 0.25 mL French mini straw contained 60 million alive sperm and froze according to the protocol. The ND1 mtDNA amplification of samples was carried out Polymerase Chain Reaction machine, followed by DNA sequencing using the Sanger method. Meanwhile, the phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software. The results showed that the T2 group maintained the highest quality for Kacang buck post-thawed semen. There was the highest percentages of sperms viability, motility, intact plasma membrane (IPM), the lowest of malondialdehyde (MDA) concentration, sperm DNA fragmentation (SDF), the total and types of ND1 mtDNA mutation frequency. The phylogenetic tree analysis revealed that the clade of the T2 group was most closely related to the sequence reference. However, there was no correlation between the semen quality parameters (sperm viability, motility, IPM, MDA concentration, and SDF) with ND1 mtDNA mutation of post-thawed Kacang buck semen. It could be concluded that GTE was useful as an antioxidant for Kacang buck semen extender for frozen sperm.

Lack of Electron Acceptors Contributes to Redox Stress and Growth Arrest in Asparagine-Starved Sarcoma Cells.

Simple SummaryCancer cells require amino acids to grow and survive. Amino acid starvation inhibits cancer growth. This study investigates why the non-essential amino acid asparagine is important for cancer growth by examining metabolite composition in asparagine-deprived sarcoma cells compared to control cells with normal asparagine access. Our experiments show that asparagine deprivation results in an imbalance between certain antioxidants and free radicals in the cell. Chemicals which impair the regeneration of antioxidants in the cell augment the growth inhibition caused by asparagine starvation.Amino acids are integral components of cancer metabolism. The non-essential amino acid asparagine supports the growth and survival of various cancer cell types. Here, different mass spectrometry approaches were employed to identify lower aspartate levels, higher aspartate/glutamine ratios and lower tricarboxylic acid (TCA) cycle metabolite levels in asparagine-deprived sarcoma cells. Reduced nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide hydride (NADH) ratios were consistent with redirection of TCA cycle flux and relative electron acceptor deficiency. Elevated lactate/pyruvate ratios may be due to compensatory NAD+ regeneration through increased pyruvate to lactate conversion by lactate dehydrogenase. Supplementation with exogenous pyruvate, which serves as an electron acceptor, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Chemicals disrupting NAD+ regeneration in the electron transport chain further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. We speculate that reductive stress may be a major contributor to the growth arrest observed in asparagine-starved cells.

Spatiotemporal Subcellular Characterization of Absolute NADH Concentration Over the Dynamic Course of Metaphase, Anaphase, Telophase and Cytokinesis using the Phasor Approach to FLIM

Absolute concentration measurements of free and bound nicotinamide adenine dinucleotide hydride (NADH) using the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) is a powerful method for discerning metabolic robustness in living cells with subcellular resolution. Cell cycle and metabolism are at the forefront of ongoing cancer research to explore new targets for cancer therapeutics. A key feature of cancer cells is continual mitotic division. Completion of mitosis ends with the progression of cells through metaphase, anaphase, telophase and cytokinesis. Each of these phases last approximately 3 minutes, presenting a large challenge for researchers to assess metabolism at these stages using available biochemical approaches. Using the phasor FLIM paired with Digital Frequency Domain (DFD) FLIM hardware, we spatiotemporally measure the absolute concentration of NADH (a measure of metabolic robustness) and free/bound NADH ratio (a measure of metabolic mode) in individual mitochondria of cells progressing from metaphase through cytokinesis. Our timelapse FLIM measurements of HeLa cells characterize the metabolic heterogeneity of mitochondria metabolism based on spatial location at the cell equator or spindle poles during the temporal progression of cells from metaphase through cytokinesis. We observe mitochondria NADH concentration and free/bound NADH at the spindle equator and pole correlate in early metaphase, but diverge starting late metaphase to early anaphase coinciding with onset of anaphase cell elongation. Our subcellular NADH FLIM analysis results support the existence of a spatiotemporal specific mechanism for mitochondria function during the course of cell division. Supported by NIGMS P41GM103540 to EG and MD.

Characterisation of a catalytic triad and reaction selectivity in the dual mechanism of the catalyse hydride transfer in xylitol phosphate dehydrogenase

Xylitol is a high-value low-calorie sweetener used as sugar substitute in food and pharmaceutical industry. Xylitol phosphate dehydrogenase (XPDH) catalyses the conversion of d-xylulose 5-phosphate (XU5P) and d-ribulose 5-phosphate (RU5P) to xylitol and ribitol respectively in the presence of nicotinamide adenine dinucleotide hydride (NADH). Although these enzymes have been shown to produce xylitol and ribitol, there is an incomplete understanding of the mechanism of the catalytic events of these reactions and the detailed mechanism has yet to be elucidated. The main goal of this work is to analyse the conformational changes of XPDH-bound ligands such as zinc, NADH, XU5P, and RU5P to elucidate the key amino acids involved in the substrate binding. In silico modelling, comparative molecular dynamics simulations, interaction analysis and conformational study were carried out on three XPDH enzymes of the Medium-chain dehydrogenase (MDR) family in order to elucidate the atomistic details of conformational transition, especially on the open and closed state of XPDH. The analysis also revealed the possible mechanism of substrate specificity that are responsible in the catalyse hydride transfer are the residues His58 and Ser39 which would act as the proton donor for reduction of XU5P and RU5P respectively. The structural comparison and MD simulations displayed a significant difference in the conformational dynamics of the catalytic and coenzyme loops between Apo and XPDH-complexes and highlight the contribution of newly found triad residues. This study would assist future mutagenesis study and enzyme modification work to increase the catalysis efficiency of xylitol production in the industry.

Multilocus Metabarcoding of Terrestrial Leech Bloodmeal iDNA Increases Species Richness Uncovered in Surveys of Vertebrate Host Biodiversity.

Leech-derived invertebrate DNA (iDNA) has been successfully leveraged to conduct surveys of vertebrate host biodiversity across the Indo Pacific. However, this technique has been limited methodologically, typically only targeting mammalian 16S rDNA, or both 16S and vertebrate 12S rDNA for leech host determination. To improve the taxonomic richness of vertebrate host species in iDNA surveys, we re-analyze datasets from Bangladesh, Cambodia, China, and Madagascar through metabarcoding via next generation sequencing (NGS) of 12S, 16S (2 types, one designed to target mammals and the other, residual eDNA), nicotinamide adenine dinucleotide hydride dehydrogenase 2 (ND2), and cytochrome c oxidase subunit 1 (COI). With our 5 primer sets, we identify 41 unique vertebrate hosts to the species level, among 1,200 leeches analyzed, along with an additional 13 taxa to the family rank. Within our 41 taxa, we note that adding ND2 and COI loci increased species richness detection by 25%. NGS has emerged as more efficient than Sanger sequencing for large scale metabarcoding applications and, with the decline in cost of NGS, our pooled sample multilocus protocol is an attractive option for iDNA biodiversity surveys.

161 Dichloroacetate influences the mitochondrial activity of bovine oocytes impairing meiotic progression

Pyruvate is a key energy substrate for the oocyte during maturation and acquisition of developmental competence. Mitochondrial activity is also essential for oocyte competence. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that indirectly stimulates pyruvate dehydrogenase (PDH), increasing pyruvate oxidation. PDH converts pyruvate into acetyl coenzyme A (acetyl-CoA) and thereby modulates the entry of glucose-derived carbons into the tricarboxylic acid (TCA) cycle, the main ATP production pathway within the oocyte. It was reported that DCA addition to embryo culture media improves embryo development in aged mice, by enhancing mitochondrial membrane potential (MMP) and decreasing oxidative stress (McPherson et al. 2014 Fertil. Steril. 101, 1458-1466). We hypothesised that increased pyruvate metabolism through the oxidative pathway, by stimulating PDH activity with DCA, could influence in vitro oocyte maturation. The aim of this work was to evaluate the effect of different concentrations of DCA during in vitro maturation (IVM) of bovine oocytes on maturation rate and mitochondrial activity, by assessing MMP and levels of flavin adenine dinucleotide (FADH2), nicotinamide adenine dinucleotide hydride (NADH), and reactive oxygen species (ROS). Abattoir-derived bovine cumulus-oocytes complexes (COC; n=360, over 4 replicates) were in vitro-matured with 0 (Control; n=120), 0.5mM (n=120) and 5mM (n=120) of DCA. After maturation, all matured COC were denuded by mechanical pipetting and meiotic progression was assessed by Hoechst 33342 staining and MMP by MitoTracker Red CMXRos test (Thermo Fisher Scientific, Waltham, MA, USA). Moreover, FADH2 and NADH levels were evaluated by autofluorescence (Dumollard et al. Development 134, 455-465) and ROS levels by CellRox® Green test (Thermo Fisher Scientific). Data were analysed by ANOVA, and the Tukey post hoc test was used to evaluate the difference among groups. The α-level was set at 0.05. Treatment with both concentrations of DCA decreased maturation rate (86.1, 67.8, and 67.6% in 0, 0.5, and 5mM groups, respectively; P<0.05). The MMP increased in oocytes matured with the highest concentration of DCA (3.42±0.28, 4.44±0.51, and 6.32±0.89 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05). In line with this, higher levels of FADH2 (3.16±0.15, 3.96±0.24, and 3.83±0.20 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05) and NADH (3.86±0.14, 4.80±0.16, and 4.95±0.17 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05) were found in both DCA-treated groups compared with the control. Unexpectedly, ROS levels increased in the presence of DCA (0.9±0.07, 1.30±0.12, and 1.54±0.16 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05) compared with the control. These results suggest that DCA was effective in stimulating mitochondrial activity of bovine oocytes, but also resulting in increased oxidative stress that likely accounts for the decreased maturation rate. Therefore, alternative strategies should be identified for the manipulation of the oocyte metabolic profile to improve oocyte developmental competence.

NADH dehydrogenase complexI is overexpressed in incipient metastatic murine colon cancer cells.

Colon cancer is one of the most frequently occurring types of cancers in the world. Primary tumours are treated very efficiently, but the metastatic cases are known to have severe outcomes. Therefore, the aim of the present study was to obtain a greater understanding of the transformation of primary colon cancer cells into metastatic phenotypes. Small changes in protein expression provoke the metastatic phenotype transformation. More sensitive methods to detect small variations are required. A murine colon cancer cell line with metastatic characteristics in a very early phase was created in order to investigate the first steps of transformation using a murine liver metastasis model. The protein expression patterns of metastatic and non-metastatic cells were compared using the stable isotope labelling by amino acids in cell culture method in combination with mass spectrometry. Quantitative proteomics data indicated that nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase complex I was overexpressed in metastatic cells with respect to non-metastatic cells. Since the NADH dehydrogenase complex catalyses the oxidation of NADH to NAD+, the functionality of the complex was studied by measuring the amount of NADH. The results revealed that metastatic cells accumulate more NADH and reactive oxygen species. In addition, the mitochondrial membrane potential of metastatic cells was lower than that of non-metastatic cells, indicating that the activity of NADH dehydrogenase and the mitochondrial oxidative chain were decreased in metastatic cells. During the incipient transformation of primary cancer cells, NADH dehydrogenase complex I was overexpressed but then became inactive due to the Warburg effect, which inhibits mitochondrial activity. In the first step of transformation, the high energy demand required in an adverse environment is fulfilled by overexpressing components of the respiratory chain, a fact that should be considered for future anti-metastatic therapies.

Primitive Photosynthetic Architectures Based on Self-Organization and Chemical Evolution of Amino Acids and Metal Ions.

The emergence of light‐energy‐utilizing metabolism is likely to be a critical milestone in prebiotic chemistry and the origin of life. However, how the primitive pigment is spontaneously generated still remains unknown. Herein, a primitive pigment model based on adaptive self‐organization of amino acids (Cystine, Cys) and metal ions (zinc ion, Zn2+) followed by chemical evolution under hydrothermal conditions is developed. The resulting hybrid microspheres are composed of radially aligned cystine/zinc (Cys/Zn) assembly decorated with carbonate‐doped zinc sulfide (C‐ZnS) nanocrystals. The part of C‐ZnS can work as a light‐harvesting antenna to capture ultraviolet and visible light, and use it in various photochemical reactions, including hydrogen (H2) evolution, carbon dioxide (CO2) photoreduction, and reduction of nicotinamide adenine dinucleotide (NAD+) to nicotinamide adenine dinucleotide hydride (NADH). Additionally, guest molecules (e.g., glutamate dehydrogenase, GDH) can be encapsulated within the hierarchical Cys/Zn framework, which facilitates sustainable photoenzymatic synthesis of glutamate. This study helps deepen insight into the emergent functionality (conversion of light energy) and complexity (hierarchical architecture) from interaction and reaction of prebiotic molecules. The primitive pigment model is also promising to work as an artificial photosynthetic microreactor.