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Archives of Pathology & Laboratory... Sep 2013DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps,... (Review)
Review
CONTEXT
DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined.
OBJECTIVE
To demonstrate the fundamental principles of pyrosequencing.
DATA SOURCES
Salient features of pyrosequencing are demonstrated using the free software program Pyromaker ( http://pyromaker.pathology.jhmi.edu ), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results.
CONCLUSIONS
We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.
Topics: Base Sequence; DNA Mutational Analysis; DNA, Neoplasm; Diphosphates; Genotype; High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Polymerase Chain Reaction; Sequence Analysis, DNA; Software
PubMed: 23991743
DOI: 10.5858/arpa.2012-0463-RA -
International Journal of Molecular... Dec 2021Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are... (Review)
Review
Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.
Topics: Diphosphates; Humans; Phosphates; Vascular Calcification
PubMed: 34948333
DOI: 10.3390/ijms222413536 -
Nature Jun 2022All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene. This approach is fundamentally different from the biosynthesis of...
All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene. This approach is fundamentally different from the biosynthesis of short-chain (C-C) terpenes that are formed from polyisoprenyl diphosphates. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.
Topics: Ascomycota; Colletotrichum; Cyclization; Diphosphates; Squalene; Substrate Specificity; Talaromyces; Triterpenes
PubMed: 35650436
DOI: 10.1038/s41586-022-04773-3 -
Current Opinion in Rheumatology Nov 2022The aim of this study was to provide updated information on the prevalence, pathogenesis, diagnostics and therapeutics of calcinosis cutis associated with systemic... (Review)
Review
PURPOSE OF REVIEW
The aim of this study was to provide updated information on the prevalence, pathogenesis, diagnostics and therapeutics of calcinosis cutis associated with systemic sclerosis (SSc).
RECENT FINDINGS
Observational studies show ethnic and geographical differences in the prevalence of calcinosis. In addition to clinical and serological associations, biochemical studies and in-vivo models have attempted to explain theories behind its pathogenesis, including prolonged state of inflammation, mechanical stress, hypoxia and dysregulation in bone and phosphate metabolism. Long-term use of proton pump inhibitors may increase the risk for calcinosis in SSc. Few single-centre observational studies have shown mild benefit with minocycline and topical sodium thiosulfate.
SUMMARY
Calcinosis cutis is the deposition of insoluble calcium in the skin and subcutaneous tissues. It affects up to 40% of SSc patients and causes significant morbidity. Long disease duration, features of vascular dysfunction and osteoporosis have been associated with calcinosis. Altered levels of inorganic pyrophosphate and fibroblast growth factor-23 have been implicated in dysregulated phosphate metabolism that may lead to calcinosis in SSc. Plain radiography can help with diagnosis and quantifying the calcinosis burden. Surgical treatment remains the most effective therapy when feasible. At present, no medical therapies have proven efficacy in large randomized controlled trials.
Topics: Calcinosis; Calcium; Diphosphates; Humans; Minocycline; Proton Pump Inhibitors; Scleroderma, Systemic
PubMed: 35993867
DOI: 10.1097/BOR.0000000000000896 -
Cellular Microbiology Jun 2021Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular processes in eukaryotic cells. IPs and PP-IPs are highly negatively charged... (Review)
Review
Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular processes in eukaryotic cells. IPs and PP-IPs are highly negatively charged and exert their biological effects by interacting with specific protein targets. Studies performed predominantly in mammalian cells and model yeasts have shown that IPs and PP-IPs modulate target function through allosteric regulation, by promoting intra- and intermolecular stabilization and, in the case of PP-IPs, by donating a phosphate from their pyrophosphate (PP) group to the target protein. Technological advances in genetics have extended studies of IP function to microbial pathogens and demonstrated that disrupting PP-IP biosynthesis and PP-IP-protein interaction has a profound impact on pathogenicity. This review summarises the complexity of IP-mediated regulation in eukaryotes, including microbial pathogens. It also highlights examples of poor conservation of IP-protein interaction outcome despite the presence of conserved IP-binding domains in eukaryotic proteomes.
Topics: Bacteria; Diphosphates; Eukaryotic Cells; Humans; Inositol; Polyphosphates; Protein Interaction Maps; Proteome; Virulence
PubMed: 33721399
DOI: 10.1111/cmi.13325 -
Biochemical Society Transactions Feb 2016To help define the molecular basis of cellular signalling cascades, and their biological functions, there is considerable value in utilizing a high-quality chemical... (Review)
Review
To help define the molecular basis of cellular signalling cascades, and their biological functions, there is considerable value in utilizing a high-quality chemical 'probe' that has a well-defined interaction with a specific cellular protein. Such reagents include inhibitors of protein kinases and small molecule kinases, as well as mimics or antagonists of intracellular signals. The purpose of this review is to consider recent progress and promising future directions for the development of novel molecules that can interrogate and manipulate the cellular actions of inositol pyrophosphates (PP-IPs)--a specialized, 'energetic' group of cell-signalling molecules in which multiple phosphate and diphosphate groups are crammed around a cyclohexane polyol scaffold.
Topics: Animals; Diphosphates; Humans; Inositol Phosphates; Phosphotransferases (Phosphate Group Acceptor); Protein Kinase Inhibitors; Signal Transduction
PubMed: 26862205
DOI: 10.1042/BST20150184 -
Antiviral Chemistry & Chemotherapy Dec 2017In this review, our recent advances in the development of nucleoside di- and nucleoside triphosphate prodrugs is summarized. Previously, we had developed a successful... (Review)
Review
In this review, our recent advances in the development of nucleoside di- and nucleoside triphosphate prodrugs is summarized. Previously, we had developed a successful membrane-permeable pronucleotide system for the intracellular delivery of nucleoside monophosphates as well, the so-called cycloSal-approach. In contrast to that work in which the delivery is initiated by a chemically driven hydrolysis reaction, for the di- and triphosphate delivery, an enzymatic trigger mechanism involving (carboxy)esterases had to be used. The other features of the new pronucleotide approaches are: (i) lipophilic modification was restricted to the terminal phosphate group leaving charges at the internal phosphate moieties and (ii) appropriate lipophilicity is introduced by long aliphatic residues within the bipartite prodrug moiety. The conceptional design of the di- and triphosphate prodrug systems will be described and the chemical synthesis, the hydrolysis properties, a structure-activity relationship and antiviral activity data will be discussed as well. The advantage of these new approaches is that all phosphorylation steps from the nucleoside analogue into the bioactive nucleoside triphosphate form can be bypassed in the case of the triphosphate prodrugs. Moreover, enzymatic processes like the deamination of nucleosides or nucleoside monophosphates which lead to catabolic clearance of the potential antivirally active compound can be avoided by the delivery of the higher phosphorylated nucleotides.
Topics: Antiviral Agents; Diphosphates; Humans; Microbial Sensitivity Tests; Nucleosides; Polyphosphates; Prodrugs; Viruses
PubMed: 29096525
DOI: 10.1177/2040206617738656 -
Brazilian Journal of Microbiology :... Sep 2021Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the...
Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the stringent response. This study reported the extraction of ppGpp from the Gram-positive bacteria Clavibacter michiganensis through methods using formic acid, lysozyme, or methanol. Following extraction, ppGpp was detected through ultra-high-performance liquid chromatography (UHPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methanol method showed the highest extraction efficiency for ppGpp among the three methods tested. C. michiganensis cells in exponential growth phase was induced in amino acid starvation by serine hydroxamate (SHX) and used for ppGpp extraction and detection. When using the methanol extraction method, the results showed that ppGpp concentrations in SHX-treated samples were 15.645 nM, 17.656 nM, 20.372 nM, and 19.280 nM at 0 min, 15 min, 30 min and 1 h, respectively, when detected using LC-MS/MS. This is the first report on ppGpp extraction and detection in Clavibacter providing a new idea and approach for nucleotide detection and extraction in bacteria.
Topics: Amino Acids; Chromatography, Liquid; Clavibacter; Diphosphates; Guanosine Tetraphosphate; Methanol; Tandem Mass Spectrometry
PubMed: 33837930
DOI: 10.1007/s42770-021-00488-1 -
The Plant Journal : For Cell and... Sep 2022The THIAMIN REQUIRING2 (TH2) protein comprising a mitochondrial targeting peptide followed by a transcription enhancement A and a haloacid dehalogenase domain is a...
The THIAMIN REQUIRING2 (TH2) protein comprising a mitochondrial targeting peptide followed by a transcription enhancement A and a haloacid dehalogenase domain is a thiamin monophosphate (TMP) phosphatase in the vitamin B1 biosynthetic pathway. The Arabidopsis th2-3 T-DNA insertion mutant was chlorotic and deficient in thiamin diphosphate (TDP). Complementation assays confirmed that haloacid dehalogenase domain alone was sufficient to rescue the th2-3 mutant. In pTH2:TH2-GFP/th2-3 complemented plants, the TH2-GFP was localized to the cytosol, mitochondrion, and nucleus, indicating that the vitamin B1 biosynthetic pathway extended across multi-subcellular compartments. Engineered TH2-GFP localized to the cytosol, mitochondrion, nucleus, and chloroplast, could complement the th2 mutant. Together, these results highlight the importance of intracellular TMP and thiamin trafficking in vitamin B1 biosynthesis. In an attempt to enhance the production of thiamin, we created various constructs to overexpress TH2-GFP in the cytosol, mitochondrion, chloroplast, and nucleus. Unexpectedly, overexpressing TH2-GFP resulted in an increase rather than a decrease in TMP. While studies on th2 mutants support TH2 as a TMP phosphatase, analyses of TH2-GFP overexpression lines implicating TH2 may also function as a TDP phosphatase in planta. We propose a working model that the TMP/TDP phosphatase activity of TH2 connects TMP, thiamin, and TDP into a metabolic cycle. The TMP phosphatase activity of TH2 is required for TDP biosynthesis, and the TDP phosphatase activity of TH2 may modulate TDP homeostasis in Arabidopsis.
Topics: Arabidopsis; DNA-Binding Proteins; Diphosphates; Homeostasis; Phosphoric Monoester Hydrolases; Thiamine; Thiamine Pyrophosphate
PubMed: 35791282
DOI: 10.1111/tpj.15895 -
The Journal of Antibiotics Jul 2016Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the... (Review)
Review
Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.
Topics: Aspartate Carbamoyltransferase; Biocatalysis; Cyclization; Diphosphates; Geranyltranstransferase; Hydrolases; Intramolecular Lyases; Isomerases; L-Serine Dehydratase; Phosphates; Terpenes
PubMed: 27072285
DOI: 10.1038/ja.2016.39