New Processes for Ionizing Nonvolatile Compounds in Mass Spectrometry: The Road of Discovery to Current State-of-the-Art.

This covers discovery and mechanistic aspects as well as initial applications of novel ionization processes for use in mass spectrometry that guided us in a series of subsequent discoveries, instrument developments, and commercialization. matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source the use of a laser, high voltages, or any other added energy was simply unbelievable, at first. Individually and as a whole, the various discoveries and inventions started to paint, , an exciting new picture and outlook in mass spectrometry from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence.

Decapping activators Edc3 and Scd6 act redundantly with Dhh1 in post-transcriptional repression of starvation-induced pathways.

Degradation of many yeast mRNAs involves decapping by the Dcp1:Dcp2 complex. Previous studies on decapping activators Edc3 and Scd6 suggested their limited roles in mRNA decay. RNA-seq analysis of mutants lacking one or both proteins revealed that Scd6 and Edc3 have largely redundant activities in targeting numerous mRNAs for degradation that are masked in the single mutants.

Upregulation of the AMPK-FOXO1-PDK4 pathway is a primary mechanism of pyruvate dehydrogenase activity reduction in tafazzin-deficient cells.

Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified.

Insights into the roles of inositol hexakisphosphate kinase 1 (IP6K1) in mammalian cellular processes.

Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues.

Upregulation of the AMPK-FOXO1-PDK4 pathway is a primary mechanism of pyruvate dehydrogenase activity reduction and leads to increased glucose uptake in tafazzin-deficient cells.

Barth syndrome (BTHS) is a rare disorder caused by mutations in the gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1).

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle.

Anomalous peroxidase activity of cytochrome c is the primary pathogenic target in Barth syndrome.

Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL-cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism.

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase.

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity.

mRNA decapping activators Pat1 and Dhh1 regulate transcript abundance and translation to tune cellular responses to nutrient availability.

We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors. Although the Environmental Stress Response (ESR) is activated in dhh1Δ and pat1Δ mutants, hundreds of non-ESR transcripts are elevated in a manner indicating cumulative repression by Pat1 and Dhh1 in wild-type cells. These mRNAs show both reduced decapping and diminished transcription in the mutants, indicating that impaired mRNA turnover drives transcript derepression in cells lacking Dhh1 or Pat1.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in Δ cells that appears to result directly from impaired decapping rather than elevated transcription.

Cardiolipin metabolism regulates expression of muscle transcription factor MyoD1 and muscle development.

The mitochondrial phospholipid cardiolipin (CL) is critical for numerous essential biological processes, including mitochondrial dynamics and energy metabolism. Mutations in the CL remodeling enzyme TAFAZZIN cause Barth syndrome, a life-threatening genetic disorder that results in severe physiological defects, including cardiomyopathy, skeletal myopathy, and neutropenia. To study the molecular mechanisms whereby CL deficiency leads to skeletal myopathy, we carried out transcriptomic analysis of the TAFAZZIN-knockout (TAZ-KO) mouse myoblast C2C12 cell line.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide.

Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of kinase IP6K1 and repression of myo-inositol-3-P synthase.

Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-inositol-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme.

Isoforms of the transcriptional cofactor SIN3 differentially regulate genes necessary for energy metabolism and cell survival.

The SIN3 scaffolding protein is a conserved transcriptional regulator known to fine-tune gene expression. In Drosophila, there are two major isoforms of SIN3, SIN3 220 and SIN3 187, which each assemble into multi-subunit histone modifying complexes. The isoforms have distinct developmental expression patterns and non-redundant functions.

Current Knowledge on the Role of Cardiolipin Remodeling in the Context of Lipid Oxidation and Barth Syndrome.

Barth syndrome (BTHS, OMIM 302060) is a genetic disorder caused by variants of the gene (G 4.5, OMIM 300394). This debilitating disorder is characterized by cardio- and skeletal myopathy, exercise intolerance, and neutropenia.

The paradoxical role of inositol in cancer: a consequence of the metabolic state of a tumor.

Inositol is an essential nutrient, obtained either by uptake from the environment or by de novo synthesis from glucose. Inositol and its derivatives exhibit tumor-suppressive effects, potentially mediated by inhibition of the ERK-MAPK or PI3K-Akt pathways. Accordingly, many cancers have been documented to silence expression of the ISYNA1 gene, which encodes the rate-limiting enzyme of inositol synthesis.

Inositol depletion regulates phospholipid metabolism and activates stress signaling in HEK293T cells.

Inositol plays a significant role in cellular function and signaling. Studies in yeast have demonstrated an "inositol-less death" phenotype, suggesting that inositol is an essential metabolite. In yeast, inositol synthesis is highly regulated, and inositol levels have been shown to be a major metabolic regulator, with its abundance affecting the expression of hundreds of genes.

NAD supplementation improves mitochondrial performance of cardiolipin mutants.

Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-β-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function.

Studying Lipid-Related Pathophysiology Using the Yeast Model.

, commonly known as baker's yeast, is one of the most comprehensively studied model organisms in science. Yeast has been used to study a wide variety of human diseases, and the yeast model system has proved to be an especially amenable tool for the study of lipids and lipid-related pathophysiologies, a topic that has gained considerable attention in recent years. This review focuses on how yeast has contributed to our understanding of the mitochondrial phospholipid cardiolipin (CL) and its role in Barth syndrome (BTHS), a genetic disorder characterized by partial or complete loss of function of the CL remodeling enzyme tafazzin.

Cardiolipin function in the yeast S. cerevisiae and the lessons learned for Barth syndrome.

Cardiolipin (CL) is the signature phospholipid (PL) of mitochondria and plays a pivotal role in mitochondrial and cellular function. Disruption of the CL remodeling gene tafazzin (TAZ) causes the severe genetic disorder Barth syndrome (BTHS). Our current understanding of the function of CL and the mechanism underlying the disease has greatly benefited from studies utilizing the powerful yeast model Saccharomyces cerevisiae.

The Role of the UPR Pathway in the Pathophysiology and Treatment of Bipolar Disorder.

Bipolar disorder (BD) is a mood disorder that affects millions worldwide and is associated with severe mood swings between mania and depression. The mood stabilizers valproate (VPA) and lithium (Li) are among the main drugs that are used to treat BD patients. However, these drugs are not effective for all patients and cause serious side effects.

Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes.

A myo-inositol bioassay utilizing an auxotrophic strain of S. cerevisiae.

Myo-inositol is a six‑carbon sugar that is essential for the growth of mammalian cells and must be obtained through either extracellular uptake or de novo biosynthesis. The physiological importance of myo-inositol stems from its incorporation into phosphoinositides and inositol phosphates, which serve a variety of signaling, regulatory, and structural roles in cells. To study myo-inositol metabolism and function, it is essential to have a reliable method for assaying myo-inositol levels.

Valproate inhibits mitochondrial bioenergetics and increases glycolysis in Saccharomyces cerevisiae.

The widely used mood stabilizer valproate (VPA) causes perturbation of energy metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. To gain insight into these mechanisms, we determined the effects of VPA on energy metabolism in yeast. VPA treatment increased levels of glycolytic intermediates, increased expression of glycolysis genes, and increased ethanol production.