526 results on '"AlkB"'
Search Results
2. The AlkB Homolog SlALKBH10B Negatively Affects Drought and Salt Tolerance in Solanum lycopersicum.
- Author
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Shen, Hui, Zhou, Ying, Liao, Changguang, Xie, Qiaoli, Chen, Guoping, Hu, Zongli, and Wu, Ting
- Subjects
- *
DROUGHT tolerance , *TOMATOES , *ARABIDOPSIS thaliana , *SALT tolerance in plants , *GENE expression , *ALKYLATING agents , *ABSCISIC acid - Abstract
ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N6-methyladenosine (m6A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
3. Kinetic Studies on the 2-Oxoglutarate/Fe(II)-Dependent Nucleic Acid Modifying Enzymes from the AlkB and TET Families
- Author
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Zhiyuan Peng, Jian Ma, Christo Z. Christov, Tatyana Karabencheva-Christova, Nicolai Lehnert, and Deyu Li
- Subjects
kinetics ,2-OG-dependent enzyme ,AlkB ,ALKBH protein ,FTO ,TET ,Biochemistry ,QD415-436 - Abstract
Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field.
- Published
- 2023
- Full Text
- View/download PDF
4. Kinetic Studies on the 2-Oxoglutarate/Fe(II)-Dependent Nucleic Acid Modifying Enzymes from the AlkB and TET Families.
- Author
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Peng, Zhiyuan, Ma, Jian, Christov, Christo Z., Karabencheva-Christova, Tatyana, Lehnert, Nicolai, and Li, Deyu
- Subjects
NUCLEIC acids ,KETOGLUTARIC acids ,EPIGENETICS ,FORMALDEHYDE ,ESCHERICHIA coli - Abstract
Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
5. The AlkB Homolog SlALKBH10B Negatively Affects Drought and Salt Tolerance in Solanum lycopersicum
- Author
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Hui Shen, Ying Zhou, Changguang Liao, Qiaoli Xie, Guoping Chen, Zongli Hu, and Ting Wu
- Subjects
AlkB ,SlALKBH10B ,abscisic acid ,drought ,salt ,tomato ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N6-methyladenosine (m6A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B.
- Published
- 2023
- Full Text
- View/download PDF
6. A novel alkane monooxygenase (alkB) clade revealed by massive genomic survey and its dissemination association with IS elements.
- Author
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Shaojing Wang, Guoqiang Li, Zitong Liao, Tongtong Liu, and Ting Ma
- Subjects
MONOOXYGENASES ,ALKANES ,NUCLEOTIDE sequence ,FOSSIL fuels ,PETROLEUM ,DNA-binding proteins - Abstract
Background. Alkanes are important components of fossil energy, such as crude oil. The alkane monooxygenase encoded by alkB gene performs the initial step of alkane degradation under aerobic conditions. The alkB gene is well studied due to its ubiquity as well as the availability of experimentally functional evidence. The alkBFGHJKL and alkST clusters are special kind of alkB-type alkane hydroxylase system, which encode all proteins necessary for converting alkanes into corresponding fatty acids. Methods. To explore whether the alkBFGHJKL and alkST clusters were widely distributed, we performed a large-scale analysis of isolate and metagenome assembled genome data (>390,000 genomes) to identify these clusters, together with distributions of corresponding taxonomy and niches. The set of alk-genes (including but not limited to alkBGHJ) located near each other on a DNA sequence was defined as an alk-gene cluster in this study. The alkB genes with alkGHJ located nearby on a DNA sequence were picked up for the investigation of putative alk-clusters. Results. Atotal of 120 alk-gene clusters were found in 117 genomes. All the 117 genomes are from strains located only in - and -proteobacteria. The alkB genes located in alk-gene sets were clustered into a deeply branched mono-clade. Further analysis showed similarity organization types of alk-genes were observed within closely related species. Although a large number of IS elements were observed nearby, they did not lead to the wide spread of the alk-gene cluster. The uneven distribution of these elements indicated that there might be other factors affecting the transmission of alk-gene clusters. Conclusions. We conducted systematic bioinformatics research on alk-genes located near each other on a DNA sequence. This benchmark dataset of alk-genes can provide base line for exploring its evolutional and ecological importance in future studies. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
7. Lack of mismatch repair enhances resistance to methylating agents for cells deficient in oxidative demethylation.
- Author
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Gutierrez R, Chan AYS, Lai SWT, Itoh S, Lee DH, Sun K, Battad A, Chen S, O'Connor TR, and Shuck SC
- Subjects
- Humans, Demethylation, DNA Mismatch Repair, AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase metabolism, AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase genetics, MutL Protein Homolog 1 metabolism, MutL Protein Homolog 1 genetics, AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase metabolism, AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase genetics
- Abstract
The human alkylation B (AlkB) homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair enzymes that remove 1-methyladenine (1meA) and 3-methylcytosine (3meC) lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1
ko ALKBH3ko cells showed enhanced resistance toward SN 1- and SN 2-type methylating agents, whereas MLH1ko ALKBH2ko cells were only resistant to SN 1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH2ko 3ko ) rendered cells sensitive to SN 1- and SN 2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH2ko 3ko cells have an ATR-dependent arrest at the G2 /M checkpoint, increased apoptotic signaling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1ko ALKBH2ko 3ko cells. Finally, in MLH1ko ALKBH2ko 3ko cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF
8. High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)
- Author
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Hrabeta-Robinson, Eva, Marcus, Erin, Cozen, Aaron E, Phizicky, Eric M, and Lowe, Todd M
- Subjects
Biological Sciences ,Bioinformatics and Computational Biology ,Genetics ,AlkB Enzymes ,Animals ,Gene Library ,High-Throughput Nucleotide Sequencing ,Humans ,Methylation ,RNA ,RNA ,Transfer ,Sequence Analysis ,RNA ,RNA Sequencing ,Transfer RNA ,AlkB ,RNA demethylation ,N-1-methyladenosine (m(1)A) ,N-3-methylcytidine (m(3)C) ,N-1-methylguanosine (m(1)G) ,N 1-methyladenosine ,N 1-methylguanosine ,N 3-methylcytidine ,Other Chemical Sciences ,Biochemistry and Cell Biology ,Developmental Biology ,Biochemistry and cell biology ,Medicinal and biomolecular chemistry - Abstract
N 1-methyladenosine (m1A), N 3-methylcytidine (m3C), and N 1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.
- Published
- 2017
9. An Overview of the Electron-Transfer Proteins That Activate Alkane Monooxygenase (AlkB).
- Author
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Williams, Shoshana C. and Austin, Rachel Narehood
- Subjects
FERREDOXIN-NADP reductase ,MONOOXYGENASES ,OPERONS ,CARBON cycle ,ALKANES ,PROTEINS ,FERREDOXINS - Abstract
Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron–sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
10. Genome-wide sequence identification and expression analysis of N6-methyladenosine demethylase in sugar beet (Beta vulgaris L.) under salt stress.
- Author
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Jie Cui, Junli Liu, Junliang Li, Dayou Cheng, and Cuihong Dai
- Subjects
DEMETHYLASE ,RNA modification & restriction ,SUGAR beets ,BEETS ,SALT ,ARABIDOPSIS thaliana ,ABIOTIC stress - Abstract
In eukaryotes, N
6 -methyladenosine (m6A) is the most abundant and highly conserved RNA modification. In vivo, m6 A demethylase dynamically regulates the m6 A level by removing the m6 A marker where it plays an important role in plant growth, develop- ment and response to abiotic stress. The confirmed m6A demethylases in Arabidopsis thaliana include ALKBH9B and ALKBH10B, both belonging to the ALKB family. In this study, BvALKB family members were identified in sugar beet genome-wide database, and their conserved domains, gene structures, chromosomal locations, phylogeny, conserved motifs and expression of BvALKB genes were analyzed. Almost all BvALKB proteins contained the conserved domain of 2OG-Fe II-Oxy. Phylogenetic analysis suggested that the ten proteins were clustered into five groups, each of which had similar motifs and gene structures. Three Arabidopsis m6A demethylase-homologous proteins (BvALKBH6B, BvALKBH8B and BvALKBH10B) were of particular interest in our study. Expression profile analysis showed that almost all genes were up-regulated or down-regulated to varying degrees under salt stress. More specifically, BvALKBH10B homologous to AtALKBH10B was significantly up-regulated, suggesting that the transcriptional activity of this gene is responsive to salt stress. This study provides a theoretical basis for further screening of m6A demethylase in sugar beet, and also lays a foundation for studying the role of ALKB family proteins in growth, development and response to salinity stress. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
11. An Overview of the Electron-Transfer Proteins That Activate Alkane Monooxygenase (AlkB)
- Author
-
Shoshana C. Williams and Rachel Narehood Austin
- Subjects
alkane monooxygenase ,AlkB ,rubredoxin ,fusion-protein ,hydrocarbon oxidation ,Microbiology ,QR1-502 - Abstract
Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron–sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research.
- Published
- 2022
- Full Text
- View/download PDF
12. Genome-wide sequence identification and expression analysis of N6-methyladenosine demethylase in sugar beet (Beta vulgaris L.) under salt stress
- Author
-
Jie Cui, Junli Liu, Junliang Li, Dayou Cheng, and Cuihong Dai
- Subjects
Sugar beet ,N6-methyladenosine ,Demethylase ,ALKB ,Salt stress ,Bioinformatics ,Medicine ,Biology (General) ,QH301-705.5 - Abstract
In eukaryotes, N6-methyladenosine (m6A) is the most abundant and highly conserved RNA modification. In vivo, m6A demethylase dynamically regulates the m6A level by removing the m6A marker where it plays an important role in plant growth, development and response to abiotic stress. The confirmed m6A demethylases in Arabidopsis thaliana include ALKBH9B and ALKBH10B, both belonging to the ALKB family. In this study, BvALKB family members were identified in sugar beet genome-wide database, and their conserved domains, gene structures, chromosomal locations, phylogeny, conserved motifs and expression of BvALKB genes were analyzed. Almost all BvALKB proteins contained the conserved domain of 2OG-Fe II-Oxy. Phylogenetic analysis suggested that the ten proteins were clustered into five groups, each of which had similar motifs and gene structures. Three Arabidopsis m6A demethylase-homologous proteins (BvALKBH6B, BvALKBH8B and BvALKBH10B) were of particular interest in our study. Expression profile analysis showed that almost all genes were up-regulated or down-regulated to varying degrees under salt stress. More specifically, BvALKBH10B homologous to AtALKBH10B was significantly up-regulated, suggesting that the transcriptional activity of this gene is responsive to salt stress. This study provides a theoretical basis for further screening of m6A demethylase in sugar beet, and also lays a foundation for studying the role of ALKB family proteins in growth, development and response to salinity stress.
- Published
- 2022
- Full Text
- View/download PDF
13. DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus.
- Author
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Osorio-Concepción, Macario, Lax, Carlos, Navarro, Eusebio, Nicolás, Francisco E., and Garre, Victoriano
- Subjects
- *
DNA methylation , *ADENINE , *MUCORMYCOSIS , *MACROPHAGES - Abstract
The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
14. A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in Escherichia coli.
- Author
-
Silas, Yardena, Singer, Esti, Das, Koyeli, Lehming, Norbert, and Pines, Ophry
- Subjects
- *
DNA repair , *DNA damage , *DNA ligases , *DOUBLE-strand DNA breaks , *KREBS cycle , *ESCHERICHIA coli , *COMMERCIAL products - Abstract
Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC. Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alphaketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
15. Bacterial Community Response to Hydrocarbon Contamination in Soils and Marine Sediments: A Critical Review of Case Studies
- Author
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Vandera, Elpiniki, Koukkou, Anna I., Cravo-Laureau, Cristiana, editor, Cagnon, Christine, editor, Lauga, Béatrice, editor, and Duran, Robert, editor
- Published
- 2017
- Full Text
- View/download PDF
16. Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification.
- Author
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Warren, Jessica M., Salinas-Giegé, Thalia, Hummel, Guillaume, Coots, Nicole L., Svendsen, Joshua M., Brown, Kristen C., Drouard, Laurence, and Sloan, Daniel B.
- Subjects
TRANSFER RNA ,GENE expression ,RNA sequencing ,POLYMERASE chain reaction ,ARABIDOPSIS thaliana - Abstract
Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be 'hot spots' for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
17. DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus
- Author
-
Macario Osorio-Concepción, Carlos Lax, Eusebio Navarro, Francisco E. Nicolás, and Victoriano Garre
- Subjects
mucormycosis ,AlkB ,demethylase ,virulence ,dimorphism ,protein kinase A ,Biology (General) ,QH301-705.5 - Abstract
The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.
- Published
- 2021
- Full Text
- View/download PDF
18. Sequence Dependent Repair of 1,N6-Ethenoadenine by DNA Repair Enzymes ALKBH2, ALKBH3, and AlkB
- Author
-
Rui Qi, Ke Bian, Fangyi Chen, Qi Tang, Xianhao Zhou, and Deyu Li
- Subjects
mutational spectra ,mutational signatures ,DNA repair ,εA ,AlkB ,ALKBH2/3 ,Organic chemistry ,QD241-441 - Abstract
Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N6-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5′/3′ flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.
- Published
- 2021
- Full Text
- View/download PDF
19. High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias
- Author
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Sander Plessers, Vincent Van Deuren, Rob Lavigne, and Johan Robben
- Subjects
phage display ,AlkB ,FTO ,Illumina sequencing ,Oxford nanopore sequencing ,parasitic enrichment ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.
- Published
- 2021
- Full Text
- View/download PDF
20. The AlkB Homolog SlALKBH10B Negatively Affects Drought and Salt Tolerance in Solanum lycopersicum .
- Author
-
Shen H, Zhou Y, Liao C, Xie Q, Chen G, Hu Z, and Wu T
- Subjects
- Salt Tolerance genetics, Droughts, Phylogeny, Abscisic Acid, Escherichia coli, AlkB Enzymes, Mixed Function Oxygenases, Arabidopsis, Solanum lycopersicum genetics, Escherichia coli Proteins
- Abstract
ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N
6 -methyladenosine (m6 ), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato ( Arabidopsis thaliana ), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato ( Solanum lycopersicum ) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B , we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B .- Published
- 2023
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21. Detection of chemical exchange in methyl groups of macromolecules.
- Author
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Gill, Michelle L., Hsu, Andrew, and Palmer, Arthur G.
- Subjects
METHYL groups ,MACROMOLECULES ,NUCLEAR magnetic resonance ,MOLECULAR dynamics - Abstract
The zero- and double-quantum methyl TROSY Hahn-echo and the methyl
1 H–1 H dipole–dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for E. coli ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of E. coli AlkB. [ABSTRACT FROM AUTHOR]- Published
- 2019
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22. A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3
- Author
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Yuko Ueda, Kaori Kitae, Ikumi Ooshio, Yasuyuki Fusamae, Megumi Kawaguchi, Kentaro Jingushi, Kazuo Harada, Kazumasa Hirata, and Kazutake Tsujikawa
- Subjects
AlkB ,ALKBH3 ,Demethylation ,3-methylcytosine ,RT-PCR ,Biology (General) ,QH301-705.5 ,Biochemistry ,QD415-436 - Abstract
Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.
- Published
- 2016
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23. Role of ALKBH1 in the Core Transcriptional Network of Embryonic Stem Cells
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Rune Ougland, Ida Jonson, Marivi Nabong Moen, Gaute Nesse, Gry Asker, Arne Klungland, and Elisabeth Larsen
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AlkB ,AlkB homologs ,ALKBH1 ,NANOG ,SOX2 ,Epigenetics ,Histone demethylation ,Embryonic stem cells ,Pluripotency ,Histone H2A ,Physiology ,QP1-981 ,Biochemistry ,QD415-436 - Abstract
Background/Aims: ALKBH1, an AlkB homologue in the 2-oxoglutarate and Fe2+ dependent hydroxylase family, is a histone dioxygenase that removes methyl groups from histone H2A. Studies of transgenic mice lacking Alkbh1 reveal that most Alkbh1-/- embryos die during embryonic development. Embryonic stem cells (ESCs) derived from these mice have prolonged expression of pluripotency markers and delayed induction of genes involved in neural differentiation, indicating that ALKBH1 is involved in regulation of pluripotency and differentiation. The aim of this study was to further investigate the role ALKBH1 in early development. Methods: Double-filter methods for nitrocellulose-filter binding, dot blot, enzyme-linked immunosorbent assay (ELISA), immonocytochemistry, cell culture and differentiation of mouse ESCs, Co-IP and miRNA analysis. Results: We found that SOX2 and NANOG bind the ALKBH1 promoter, and we identified protein-protein interactions between ALKBH1 and these core transcription factors of the pluripotency network. Furthermore, lack of ALKBH1 affected the expression of developmentally important miRNAs, which are involved in the regulation of NANOG, SOX2 and neural differentiation. Conclusion: Our results suggest that ALKBH1 interacts with the core transcriptional pluripotency network of ESCs and is involved in regulation of pluripotency and differentiation.
- Published
- 2016
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24. Escherichia coli AlkB and single-stranded DNA binding protein SSB interaction explored by Molecular Dynamics Simulation.
- Author
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Mohan, Monisha, Pandya, Vishal, and Anindya, Roy
- Subjects
- *
ESCHERICHIA coli , *DNA-binding proteins , *METHYLCYTOSINE , *DEMETHYLATION , *DNA repair - Abstract
Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia Coli ( E.coli ) DNA repair enzyme AlkB removes methyl adducts including 1-methyladenine and 3-methylcytosine present in DNA by oxidative demethylation from single-stranded DNA (ssDNA). E. coli single-stranded DNA binding protein (SSB) selectively binds ssDNA in a sequence-independent manner. We have recently shown that AlkB can repair methyl adduct present in SSB-coated ssDNA. In this study, we aimed to elucidate details of AlkB-mediated DNA repair of SSB-bound DNA substrate. Therefore, we generated a structural model of AlkB-SSB-ssDNA and using Molecular Dynamics simulation analysis we show that flexibility of SSB-bound DNA allows AlkB to bind in multiple ways. Our docking analysis of AlkB-SSB-ssDNA structure revealed that the Cyt109 base is present in the hydrophobic cavity of AlkB active site pocket. The characterization of AlkB-SSB interaction pattern would likely to help in understanding the mode of alkylated DNA adduct recognition by AlkB. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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25. DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in Saccharomyces cerevisiae.
- Author
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Deepa, Akula, Naveena, Kodipelli, and Anindya, Roy
- Subjects
- *
SACCHAROMYCES cerevisiae , *DNA repair , *DIOXYGENASES , *IRON deficiency , *TRANSCRIPTION factors - Abstract
Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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26. Microaerophilic alkane degradation in Pseudomonas extremaustralis: a transcriptomic and physiological approach.
- Author
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Tribelli, Paula M., Rossi, Leticia, Ricardi, Martiniano M., Gomez-Lozano, Maria, Molin, Søren, Raiger Iustman, Laura J., and Lopez, Nancy I.
- Subjects
- *
DIESEL fuels , *HYDROCARBONS , *ALKANES , *AROMATIC compounds , *PSEUDOMONAS - Abstract
Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13-C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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27. Expression and molecular profiles of the AlkB family in ovarian serous carcinoma
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Shuangshuang Zeng, Zhijie Xu, Geting Wu, Yuanliang Yan, Juanni Li, Yuan Cai, and Bi Peng
- Subjects
Aging ,AlkB ,Biology ,Chemokine receptor ,Immune system ,AlkB family ,infiltrating immune cells ,Databases, Genetic ,Overall survival ,Humans ,Ovarian Neoplasms ,Gene Expression Profiling ,AlkB Enzymes ,Cell Biology ,Methylation ,DNA Methylation ,Prognosis ,Molecular biology ,Cystadenocarcinoma, Serous ,Gene Expression Regulation, Neoplastic ,Survival Rate ,ovarian serous carcinoma ,biology.protein ,Ovarian Serous Carcinoma ,Female ,methylation ,Signal transduction ,expression profiles ,CD8 ,Research Paper - Abstract
AlkB family of Fe (II) and α-ketoglutarate-dependent dioxygenases plays essential roles in development of ovarian serous carcinoma (OV). However, the molecular profiles of AlkB family in OV have not been clarified. The results indicated that the expression of ALKBH1/3/5/8 and FTO was lower in OV patients while ALKBH2/4/6/7 expression was higher. There was a strong correlation between ALKBH5/7 and pathological stage of OV patients. Kaplan-Meier plotter revealed that OV patients with high ALKBH4 level showed longer overall survival (OS). However, patients with high levels of ALKBH5/6 and FTO showed shorter OS and progression-free survival (PFS). Genetic alterations using cBioPortal revealed that the alteration rates of FTO were the highest. We also found that the functions of AlkB family were linked to several cancer-associated signaling pathways, including chemokine receptor signaling. TIMER database indicated that the AlkB family had a strong relationship with the infiltration of six types of immune cells (macrophages, neutrophils, CD8+ T-cells, B-cells, CD4+ T-cells and dendritic cells). Next, DiseaseMeth databases revealed that the global methylation levels of ALKBH1/2/3/4/5/6/7/8 and FTO were all lower in OV patients. Thus, our findings will enhance the understanding of AlkB family in OV pathology, and provide novel insights into AlkB-targeted therapy for OV patients.
- Published
- 2021
28. Genetic analysis of low-density polyethylene degrading bacteria from plastic dump sites
- Author
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K. Vanmathi Selvi and P. Jayashree Lakshmi
- Subjects
Multidisciplinary ,biology ,AlkB ,Pseudomonas fluorescens ,Polyethylene ,Biodegradation ,biology.organism_classification ,Pseudomonas putida ,Low-density polyethylene ,chemistry.chemical_compound ,chemistry ,Biotransformation ,biology.protein ,Food science ,Bacteria - Abstract
Objective: The principle goal of this examination is to screen bacteria having the ability to debase Low density polyethylene. Bacteria were disengaged from different plastic dump locales. Methods: The secludes acquired were screened for their capacity to use polyethylene as sole carbon source in the mineral salt medium (MSM). Among the isolated strains, Pseudomonas putida and Pseudomonas fluorescens could able to utilize Low Density Polyethylene (LDPE) as carbon source. Further to the preliminary screening, gene specific primers for alkane monooxygenase was synthesized and gradient PCR was performed to determine the presence of alkane monooxygenase gene in the bacterial isolates. Results: Among the four bacteria selected, three bacteria such as Pseudomonas putida and Pseudomonas fluorescens could able to express ALKB gene. Hence, those two bacteria may produce the key enzyme alkane monoxygenase, which is a crucial enzyme for the biotransformation of many xenobiotic compounds including LDPE. Conclusion: From this investigation it is inferred that organisms local to soil can possibly degradeplastic with proper method of time. Keywords: Low density polyethylene; Biodegradation; Bacteria; alkane monoxygenase; invitro Biodegradation assay
- Published
- 2020
29. Sequence analysis of 16S rDNA, gyrB and alkB genes of plant-associated Rhodococcus species from Tunisia
- Author
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Ali Rhouma, Mark L. Gleason, Sabrine Dhaouadi, Amira Hamdane Mougou, and Chao J Wu
- Subjects
Genetics ,Rhizosphere ,Phylogenetic tree ,Sequence analysis ,AlkB ,General Medicine ,Biology ,16S ribosomal RNA ,biology.organism_classification ,Microbiology ,Phylogenetics ,biology.protein ,Phyllosphere ,Rhodococcus ,Ecology, Evolution, Behavior and Systematics - Abstract
The genusRhodococcuscontains several species with agricultural, biotechnological and ecological importance. Within this genus, many phyllosphere, rhizosphere and endosphere strains are plant growth promoting bacteria, whereas strains designated asR. fasciansare plant pathogens. In this study, we isolated 47Rhodococcusstrains from a range of herbaceous and woody plant species. Phylogenetic analysis based on 16S rDNA,gyrB andalkB genes was used to compare our strains with type strains ofRhodococcus. For most of our strains, sequence similarity of the 16S rDNA,gyrB andalkB regions to type strains ranged from 98–100 %. Results of the concatenated gene sequence comparisons identified 18 strains ofR. fasciansand three strains ofR. kroppenstedtii. The remaining strains were unclassified, and may represent novel species ofRhodococcus. Phylogenetic analysis based ongyrB sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis ofalkB sequences was unable to identify strains with orange-coloured colonies to species level.
- Published
- 2020
30. Novel Prognostic Implications of Methylated RNA and Demethylases in Resected HCC and Background Liver Tissue
- Author
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Masamichi Hayashi, Yasuhiro Kodera, Yuki Sunagawa, Suguru Yamada, Goro Nakayama, Nobuhiko Nakagawa, Fuminori Sonohara, Masahiko Koike, Raju Kandimalla, Yoshikuni Inokawa, Chie Tanaka, Mitsuro Kanda, Hideki Takami, and Katsuhito Tanaka
- Subjects
Adult ,Male ,Cancer Research ,Adenosine ,Carcinoma, Hepatocellular ,medicine.medical_treatment ,AlkB ,Alpha-Ketoglutarate-Dependent Dioxygenase FTO ,Kaplan-Meier Estimate ,Methylation ,03 medical and health sciences ,0302 clinical medicine ,Liver tissue ,medicine ,Humans ,Aged ,Proportional Hazards Models ,Aged, 80 and over ,biology ,business.industry ,Liver Neoplasms ,Significant difference ,AlkB Homolog 5, RNA Demethylase ,RNA ,General Medicine ,Middle Aged ,Prognosis ,medicine.disease ,Survival Analysis ,digestive system diseases ,Gene Expression Regulation, Neoplastic ,Liver ,Oncology ,030220 oncology & carcinogenesis ,Hepatocellular carcinoma ,Multivariate Analysis ,biology.protein ,Cancer research ,Demethylase ,Female ,Cancer development ,Hepatectomy ,business - Abstract
BACKGROUND/AIM N6-Methyladenosine (m6A), the most abundant internal modification of RNA, plays a critical role in cancer development. However, the clinical implications of m6A in hepatocellular carcinoma (HCC) remain unclear. MATERIALS AND METHODS We analyzed 177 HCC and paired noncancerous liver tissues from patients who underwent hepatectomy according to global m6A quantification and expression of m6A demethylases fat mass and obesity-associated protein (FTO) and alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5). RESULTS The global m6A quantification revealed no significant difference between HCC and non-cancerous tissue. The expression of m6A demethylases FTO and ALKBH5, was significantly lower in HCC than in non-cancerous tissues (both p
- Published
- 2020
31. Microbial Consortiums of Putative Degraders of Low-Density Polyethylene-Associated Compounds in the Ocean
- Author
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Maria Pinto, Katja Klun, Zihao Zhao, Gerhard J. Herndl, and Eugen Libowitzky
- Subjects
biology ,Chemistry ,Physiology ,Microorganism ,fungi ,AlkB ,Biofilm ,Biodegradation ,biology.organism_classification ,Biochemistry ,Microbiology ,Computer Science Applications ,Low-density polyethylene ,Metagenomics ,Environmental chemistry ,Modeling and Simulation ,biology.protein ,Genetics ,Energy source ,Molecular Biology ,Bacteria ,geographic locations ,Ecology, Evolution, Behavior and Systematics - Abstract
Polyethylene (PE) is one of the most abundant plastics in the ocean. The development of a biofilm on PE in the ocean has been reported, yet whether some of the biofilm-forming organisms can biodegrade this plastic in the environment remains unknown. Via metagenomics analysis, we taxonomically and functionally analysed three biofilm communities using low-density polyethylene (LDPE) as their sole carbon source for two years. Several of the taxa that increased in relative abundance over time were closely related to known degraders of alkane and other hydrocarbons. Alkane degradation has been proposed to be involved in PE degradation, and most of the organisms increasing in relative abundance over time harboured genes encoding proteins essential in alkane degradation, such as the genes alkB and CYP153, encoding an alkane monooxygenase and a cytochrome P450 alkane hydroxylase. Weight loss of PE sheets when incubated with these communities and chemical and electron microscopic analyses provided evidence for alteration of the PE surface over time. Taken together, these results provide evidence for the utilization of LDPE-associated compounds by the prokaryotic communities. This study identifies a group of genes potentially involved in the degradation of the LDPE polymeric structure and/or associated plastic additives in the ocean and describes a phylogenetically diverse community of plastic biofilm-dwelling microbes with the potential of utilizing LDPE-associated compounds as carbon and energy source.ImportanceLow-density polyethylene (LDPE) is one of the most used plastics worldwide and a large portion of it ends up in the ocean. Very little is known about its fate in the ocean and whether it can be biodegraded by microorganisms. By combining 2-year incubations with metagenomics, respiration measurements and LDPE surface analysis, we identified bacteria and associated genes and metabolic pathways potentially involved in LDPE biodegradation. After two years of incubation, two of the microbial communities exhibited a very similar taxonomic composition mediating changes to the LDPE pieces they were incubated with. We provide evidence that there are plastic-biofilm dwelling bacteria in the ocean, that might have the potential to degrade LDPE-associated compounds, and that alkane degradation pathways might be involved.
- Published
- 2022
32. Structure-based design of selective fat mass and obesity associated protein (FTO) inhibitors
- Author
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Afaf H. El-Sagheer, Ivanhoe K. H. Leung, Tom Brown, Michael A. McDonough, Anthony Tumber, Nok Yin Tam, Caitlin Clunie-O'Connor, Dong Zhang, Marina Demetriades, Shifali Shishodia, Wei Shen Aik, Thomas M. Leissing, Eidarus Salah, Pratheesh Maheswaran, Yi Min Ng, and Christopher J. Schofield
- Subjects
Oxygenase ,endocrine system diseases ,AlkB ,Alpha-Ketoglutarate-Dependent Dioxygenase FTO ,Antineoplastic Agents ,Crystallography, X-Ray ,010402 general chemistry ,01 natural sciences ,Article ,Mixed Function Oxygenases ,Structure-Activity Relationship ,03 medical and health sciences ,Drug Discovery ,Humans ,Binding site ,030304 developmental biology ,Demethylation ,Histone Demethylases ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,nutritional and metabolic diseases ,Substrate (chemistry) ,pathological conditions, signs and symptoms ,0104 chemical sciences ,Enzyme ,Biochemistry ,Drug Design ,biology.protein ,Nucleic acid ,Molecular Medicine - Abstract
FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.
- Published
- 2022
33. Synergetic effect of bio-photocatalytic hybrid system: g-C3N4 and Acinetobacter sp. JLS1 for enhanced degradation of C16 alkane.
- Author
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Xu, Xingjian, Zhai, Zhenhao, Li, Haiyan, Wang, Quanying, Han, Xuerong, and Yu, Hongwen
- Subjects
- *
HYDROCARBONS , *PETROLEUM , *GRAPHITE , *ACINETOBACTER , *ALKANES - Abstract
Petroleum hydrocarbons are ubiquitous in nature and generally lead to contamination. The aim of this study was to establish a bio-photocatalytic system using graphite-like carbon nitride (g-C 3 N 4 ) and petroleum-degrading bacterium Acinetobacter sp. JLS1 and evaluate its effect on C 16 alkane degradation. The successfully synthesized g-C 3 N 4 produced no obvious lethal effect to strain JLS1 until 1 g/L. C 16 alkane (0.25% v/v) degradation was investigated in the individual (photocatalytic or biocatalytic) and hybrid (bio-photocatalytic) systems. The C 16 alkane removal efficiency of the hybrid system under visible-light irradiation was 78.2% within 4 h, which was more effective than that of photocatalytic (26.8%) and biocatalytic (38.3%) systems. This positive synergetic effect between g-C 3 N 4 and strain JLS1 was due to the photocatalytic activity of g-C 3 N 4 for C 16 alkane degradation and increased porosity and permeability of bacterial membrane, thus facilitating the entry of targeted compound into bacterial cells. In the hybrid system, hexadecanoic acid, dodecanoic acid and octanoic acid were examined out in bacterial cells, which were biostransformed by strain JLS1 from initial C 16 alkane and its photocatalytic degradation products (alkanes with relative lower C atoms). In addition, alkB gene transcription was strongly upregulated by g-C 3 N 4 under visible-light irradiation, however, growth of strain JLS1 slightly improved compared to that in other tested systems, suggesting that continuous attack of photocatalytic reaction constantly impeded the repair process of bacterial cell. The established effective bio-photocatalytic system not only drew a new sight for further research but also provided a promising remediation approach for dealing with petroleum hydrocarbons. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
34. Anaerobic fixed-target serial crystallography
- Author
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Thomas M. Leissing, Pauline A. Lang, Michael A. McDonough, Bradley Davy, Robin L. Owen, Stephen B. Carr, Patrick Rabe, Julien Orlans, Pierre Aller, Anna Dirr, Allen M. Orville, Selina L. S. Storm, Christopher J. Schofield, A. Ebrahim, Danny Axford, John H. Beale, A. Butryn, DIAMOND Light source, Université Grenoble Alpes - École supérieure du professorat et de l'éducation - Académie de Grenoble (UGA ESPE Grenoble), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), ISIS Neutron and Muon Source (ISIS), STFC Rutherford Appleton Laboratory (RAL), Science and Technology Facilities Council (STFC)-Science and Technology Facilities Council (STFC), Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Wellcome Trust102593210734/Z/18/ZRoyal Society of LondonRSWF\R2\182017Deutsche Akademie fur Naturforscher Leopoldina, Germany Biotechnology and Biological Sciences Research Council (BBSRC)102593Royal Society Wolfson Fellowship RSWF\R2\182017Cancer Research UK Wellcome Investigator Award in Science 210734/Z/18/Z, Laboratoire de Modélisation et Simulation Multi Echelle (MSME), and Centre National de la Recherche Scientifique (CNRS)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Université Paris-Est Marne-la-Vallée (UPEM)
- Subjects
inorganic chemicals ,penicillin biosynthesis ,AlkB ,Isopenicillin N synthase ,010402 general chemistry ,01 natural sciences ,Biochemistry ,law.invention ,03 medical and health sciences ,law ,[CHIM]Chemical Sciences ,General Materials Science ,Sample preparation ,Reactivity (chemistry) ,serial crystallography ,lcsh:Science ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,oxygen-employing enzymes ,General Chemistry ,Condensed Matter Physics ,Research Papers ,Synchrotron ,0104 chemical sciences ,Crystallography ,Enzyme ,2-oxoglutarate/alpha-ketoglutarate oxygenases ,chemistry ,biology.protein ,anaerobic crystallization ,lcsh:Q ,Protein crystallization ,Anaerobic exercise ,2-oxoglutarate/α-ketoglutarate oxygenases - Abstract
An effective and simple-to-implement approach for anaerobic room-temperature data collection is described and demonstrated by application to dioxygen utilizing enzymes., Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the ‘sheet-on-sheet’ sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (
- Published
- 2020
35. Functional Gene Diversity of Selected Indigenous Hydrocarbon-Degrading Bacteria in Aged Crude Oil
- Author
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Chioma Bertha Ehis-Eriakha, Onyewuchi Akaranta, and Chioma Blaise Chikere
- Subjects
0301 basic medicine ,Microbiology (medical) ,education.field_of_study ,Article Subject ,biology ,030106 microbiology ,Population ,AlkB ,Bacillus cereus ,010501 environmental sciences ,Biodegradation ,biology.organism_classification ,01 natural sciences ,Microbiology ,QR1-502 ,03 medical and health sciences ,Bioremediation ,Plasmid ,biology.protein ,education ,Gene ,Bacteria ,Research Article ,0105 earth and related environmental sciences - Abstract
Crude oil pollution has consistently deteriorated all environmental compartments through the cycle of activities of the oil and gas industries. However, there is a growing need to identify microbes with catabolic potentials to degrade these pollutants. This research was conducted to identify bacteria with functional degradative genes. A crude oil-polluted soil sample was obtained from an aged spill site at Imo River, Ebubu, Komkom community, Nigeria. Bacteria isolates were obtained and screened for hydrocarbon degradation potential by turbidometry assay. Plasmid and chromosomal DNA of the potential degraders were further screened for the presence of selected catabolic genes (C230, Alma, Alkb, nahAC, and PAHRHD(GP)) and identified by molecular typing. Sixteen (16) out of the fifty (50) isolates obtained showed biodegradation activity in a liquid broth medium at varying levels. Bacillus cereus showed highest potential for this assay with an optical density of 2.450 @ 600 nm wavelength. Diverse catabolic genes resident in plasmids and chromosomes of the isolates and, in some cases, both plasmid and chromosomes of the same organism were observed. The C230 gene was resident in >50% of the microbial population tested, while other genes occurred in lower proportions with the least observed in nahAC and PAHRHD. These organisms can serve as potential bioremediation agents.
- Published
- 2020
36. Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification
- Author
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Daniel B. Sloan, Jessica M. Warren, Thalia Salinas-Giegé, Joshua M. Svendsen, Laurence Maréchal-Drouard, Nicole L. Coots, Guillaume Hummel, Kristen C. Brown, Institut de biologie moléculaire des plantes (IBMP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)
- Subjects
TRNA modification ,Arabidopsis ,AlkB ,Computational biology ,Magnoliopsida ,03 medical and health sciences ,0302 clinical medicine ,RNA, Transfer ,Gene Expression Regulation, Plant ,[SDV.BV]Life Sciences [q-bio]/Vegetal Biology ,Humans ,Arabidopsis thaliana ,Digital polymerase chain reaction ,Plastids ,Northern blot ,RNA Processing, Post-Transcriptional ,Molecular Biology ,Gene ,Protein secondary structure ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,biology ,Sequence Analysis, RNA ,High-Throughput Nucleotide Sequencing ,Cell Biology ,biology.organism_classification ,Post-transcriptional modification ,RNA, Plant ,030220 oncology & carcinogenesis ,Transfer RNA ,biology.protein ,030217 neurology & neurosurgery ,Research Paper - Abstract
Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs inArabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be “hot spots” for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.
- Published
- 2020
37. Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein
- Author
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Yu Lin, Hongsheng Wang, Jiexin Li, Nan Luo, Cheng Ming Chiang, Yuyi Ling, Zhuojia Chen, Guoyou Xie, Yanxi Peng, and Feng Chen
- Subjects
Adenosine ,AcademicSubjects/SCI00010 ,Recombinant Fusion Proteins ,CRISPR-Associated Proteins ,AlkB ,Prevotella ,Protein Engineering ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,RNA and RNA-protein complexes ,Humans ,RNA, Messenger ,030304 developmental biology ,Demethylation ,Cell Proliferation ,0303 health sciences ,Messenger RNA ,biology ,MRNA modification ,AlkB Homolog 5, RNA Demethylase ,Translation (biology) ,Protein engineering ,Oncogenes ,Fusion protein ,Cell biology ,HEK293 Cells ,030220 oncology & carcinogenesis ,biology.protein ,Narese/29 ,Demethylase ,CRISPR-Cas Systems ,5' Untranslated Regions ,HeLa Cells - Abstract
Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR–Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5–125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.
- Published
- 2020
38. Role of Structural Dynamics in Selectivity and Mechanism of Non-heme Fe(II) and 2‑Oxoglutarate-Dependent Oxygenases Involved in DNA Repair
- Author
-
Nicolai Lehnert, Rajeev Ramanan, Shobhit S. Chaturvedi, Tatyana G. Karabencheva-Christova, Christo Z. Christov, Christopher J. Schofield, and Sodiq O. Waheed
- Subjects
biology ,010405 organic chemistry ,DNA repair ,Chemistry ,Stereochemistry ,General Chemical Engineering ,AlkB ,Substrate (chemistry) ,Active site ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Molecular mechanics ,0104 chemical sciences ,3. Good health ,Nucleobase ,Molecular dynamics ,chemistry.chemical_compound ,biology.protein ,QD1-999 ,DNA ,Research Article - Abstract
AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m3C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic β-hairpin, are involved in substrate binding in AlkBH2–dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)=O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2–dsDNA and AlkB–dsDNA; in AlkB–ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein–DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism., Our study explores the effects of structural variations and the nature of the protein complexed DNA on the dynamics and mechanisms of 2-oxoglutarate- and iron-dependent DNA damage repair enzymes.
- Published
- 2020
39. Selective Electroenzymatic Oxyfunctionalization by Alkane Monooxygenase in a Biofuel Cell
- Author
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Mengwei Yuan, Christian A. Malapit, Chun You, Hui Chen, Sofiene Abdellaoui, Matthew J. Kummer, and Shelley D. Minteer
- Subjects
Hydrogenase ,Bioelectric Energy Sources ,AlkB ,Hydroxylation ,010402 general chemistry ,Methylation ,7. Clean energy ,01 natural sciences ,Catalysis ,Mixed Function Oxygenases ,Substrate Specificity ,Electron Transport ,chemistry.chemical_compound ,Safrole ,Alkanes ,Electrodes ,Demethylation ,chemistry.chemical_classification ,Alkane ,biology ,Pseudomonas putida ,010405 organic chemistry ,General Chemistry ,Monooxygenase ,biology.organism_classification ,Combinatorial chemistry ,0104 chemical sciences ,Oxygen ,Enzyme ,chemistry ,13. Climate action ,biology.protein ,Epoxy Compounds - Abstract
Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon-hydrogen (C-H) bond activation using classical chemical methods (i.e. high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Herein, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells capable of oxyfunctionalization while simultaneously producing electricity.
- Published
- 2020
40. Epigenetic Regulation of m6A Modifications in Human Cancer
- Author
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Jie Wu, Xiaoqian Qi, Wei Zhao, Shiqing Ma, Jingwen Liu, and Lina Liu
- Subjects
0301 basic medicine ,Methyltransferase ,RNA methylation ,AlkB ,Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Drug Discovery ,cancer ,Epigenetics ,Gene ,N6-methyladenosine ,lcsh:RM1-950 ,RNA ,m6A ,Cell biology ,lcsh:Therapeutics. Pharmacology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,RNA splicing ,biology.protein ,Molecular Medicine ,N6-Methyladenosine ,epigenetic - Abstract
N6-methyladenosine (m6A) is the most prevalent internal RNA modification, especially within eukaryotic messenger RNAs (mRNAs). m6A modifications of RNA regulate splicing, translocation, stability, and translation into proteins. m6A modifications are catalyzed by RNA methyltransferases, such as METTL3, METTL14, and WTAP (writers); the modifications are removed by the demethylases fat mass and obesity-associated protein (FTO) and ALKBH5 (ALKB homolog 5) (erasers); and the modifications are recognized by m6A-binding proteins, such as YTHDF domain-containing proteins and IGF2BPs (readers). Abnormal changes in the m6A levels of these genes are closely related to tumor occurrence and development. In this paper, we review the role of m6A in human cancer and summarize its prospective applications in cancer. Keywords: N6-methyladenosine, m6A, RNA methylation, epigenetic, cancer
- Published
- 2020
41. Cloning and Expression of Pseudomonas aeruginosa AlkB Gene in E. coli
- Author
-
Fadhil N. Al-Kanany and Rasha M. Othman
- Subjects
Cloning ,biology ,alkb gene ,Chemistry ,Pseudomonas aeruginosa ,AlkB ,cloning ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Molecular biology ,Microbiology ,pseudomonas aeruginosa ,QR1-502 ,biology.protein ,medicine ,Gene ,Biotechnology - Abstract
Pre identified hydrocarbons degrading bacteria were used in this study, specific primer was conducted to amplification of AlkB gene, approximately 1206bp band size of this gene for Pseudomonas aeruginosa was detected and proofed by sequence and alignment analysis with NCBI database. The AlkB gene was inserted in PET-21a(+) plasmid vector as expression vector, then transformed in BL21(DE3) competent E. coli and confirmed by colony PCR technique using the T7 promoter and T7 terminator primers. The expression of the inserted gene was checked by determined the concentration of AlkB protein for multiple periods by Bradford assay method and the SDS-polyacrylamide gel electrophoresis method was revealed band of ~46 KD molecular weight of the concerned protein. The gene amplification and cloning strategy was lay out before the practical part of the study by SnapGene software, this study was conducted to introduce cloned bacteria which facilitate the first step (key step) of alkane’s biodegradation and propose an appropriate strategy to construct genetically engineered microorganisms with multiple recombinant plasmid for enhance the degradation of the aliphatic fraction of hydrocarbon
- Published
- 2020
42. Detection of DNA Modifications by Sequence-Specific Transcription Factors
- Author
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Xiaodong Cheng, Xing Zhang, Robert Blumenthal, and Jie Yang
- Subjects
0303 health sciences ,Methyltransferase ,biology ,Chemistry ,Base pair ,DNA repair ,AlkB ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Biochemistry ,Structural Biology ,Coding strand ,biology.protein ,Epigenetics ,Molecular Biology ,030217 neurology & neurosurgery ,Polymerase ,DNA ,030304 developmental biology - Abstract
The establishment, detection, and alteration or elimination of epigenetic DNA modifications are essential to controlling gene expression ranging from bacteria to mammals. The DNA methylations occurring at cytosine and adenine are carried out by SAM-dependent methyltransferases. Successive oxidations of 5-methylcytosine (5mC) by Tet dioxygenases generate 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC) derivatives; thus, DNA elements with multiple methylation sites can have a wide range of modification states. In contrast, oxidation of N6-methyladenine by homologs of Escherichia coli AlkB removes the methyl group directly. Both Tet and AlkB enzymes are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. DNA-binding proteins decode the modification status of specific genomic regions. This article centers on two families of sequence-specific transcription factors: bZIP (basic leucine-zipper) proteins, exemplified by the AP-1 and CEBPβ recognition of 5mC; and bHLH (basic helix-loop-helix) proteins, exemplified by MAX and TCF4 recognition of 5caC. We discuss the impact of template strand DNA modification on the activities of DNA and RNA polymerases, and the varied tendencies of modifications to alter base pairing and their interactions with DNA repair enzymes.
- Published
- 2020
43. NAIL-MS reveals the repair of 2-methylthiocytidine by AlkB in E. coli
- Author
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Verena Weber, Stefanie Kellner, Dimitar Plamenov Petrov, Kirsten Jung, and Valentin F. Reichle
- Subjects
0301 basic medicine ,TRNA modification ,Science ,AlkB ,General Physics and Astronomy ,Cytidine ,Methylation ,General Biochemistry, Genetics and Molecular Biology ,Article ,Mass Spectrometry ,Mixed Function Oxygenases ,03 medical and health sciences ,Gene Knockout Techniques ,0302 clinical medicine ,RNA, Transfer ,Nucleic Acids ,Escherichia coli ,RNA Processing, Post-Transcriptional ,lcsh:Science ,tRNA Methyltransferases ,Multidisciplinary ,biology ,Chemistry ,Escherichia coli Proteins ,RNA ,Translation (biology) ,General Chemistry ,RNA modification ,TRNA Methyltransferases ,Demethylation ,RNA, Bacterial ,030104 developmental biology ,Biochemistry ,Isotope Labeling ,Transfer RNA ,Pseudomonas aeruginosa ,biology.protein ,Nucleic acid ,lcsh:Q ,030217 neurology & neurosurgery ,Chemical modification - Abstract
RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms2C) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. ms2C is only found in 2-thiocytidine (s2C) containing tRNAs, namely tRNAArgCCG, tRNAArgICG, tRNAArgUCU and tRNASerGCU at low abundances. ms2C is not formed by commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s2C containing tRNA can be methylated to carry ms2C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms2C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach., Bacterial tRNA is modified by thiolation of nucleosides. Here the authors identify 2-methylthiocytidine in bacterial tRNA using nucleic acid isotope labeling coupled mass spectrometry. Exposure to methylating agents converts 2-thiocytidine to 2-methylthiocytidine, which is repaired by demethylase AlkB in vivo.
- Published
- 2019
44. Nucleotide resolution profiling of m7G tRNA modification by TRAC-Seq
- Author
-
Shuibin Lin, Qi Liu, Richard I. Gregory, and Yi-Zhou Jiang
- Subjects
Small RNA ,TRNA modification ,Sequence analysis ,AlkB ,Computational biology ,Methylation ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,RNA, Transfer ,Animals ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Guanosine ,biology ,Sequence Analysis, RNA ,Chemistry ,High-Throughput Nucleotide Sequencing ,RNA ,Genomics ,Transfer RNA ,biology.protein ,Demethylase ,Transcriptome ,Software ,030217 neurology & neurosurgery - Abstract
Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased global mapping of 7-methylguansine (m(7)G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m(7)G TRAC-Seq involves the treatment of size-selected (
- Published
- 2019
45. Drosophila Alpha-ketoglutarate-dependent dioxygenase AlkB is involved in repair from neuronal disorders induced by ultraviolet damage
- Author
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Ibuki Ueoka, Keiko Tsuji Wakisaka, Hideki Yoshida, Ikuko Mizuta, Yuuka Muraoka, Mizuki Yamaguchi, Jo Shimizu, and Masamitsu Yamaguchi
- Subjects
Central Nervous System ,0301 basic medicine ,Xeroderma pigmentosum ,Ultraviolet Rays ,DNA damage ,AlkB ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Animals ,Eye Abnormalities ,Neurons ,chemistry.chemical_classification ,Gene knockdown ,Learning Disabilities ,General Neuroscience ,AlkB Enzymes ,Neurogenesis ,RNA ,medicine.disease ,Immunohistochemistry ,Cell biology ,030104 developmental biology ,Enzyme ,chemistry ,Gene Knockdown Techniques ,Larva ,biology.protein ,Drosophila ,Locomotion ,030217 neurology & neurosurgery ,DNA ,DNA Damage - Abstract
AlkB family proteins are enzymes that repair alkylated DNA and RNA by oxidative demethylation. Nine homologs have been identified and characterized in mammals. ALKBH1 is conserved among metazoans including Drosophila. Although the ALKBH1 mouse homolog, Alkbh1 functions in neurogenesis, it currently remains unclear whether ALKBH1 plays a role in neuronal disorders induced by ultraviolet-induced DNA damage. We herein demonstrated that the Drosophila ALKBH1 homolog, AlkB contributed to recovery from neuronal disorders induced by ultraviolet damage. The knockdown of AlkB resulted in not only learning defects but also altered crawling behavior in Drosophila larvae after ultraviolet irradiation. A molecular analysis revealed that AlkB contributed to the repair of ultraviolet-induced DNA damage in the central nervous system of larvae. Therefore, we propose that ALKBH1 plays a role in the repair of ultraviolet-induced DNA damage in central nervous system. Ultraviolet-induced DNA damage is involved in the pathogenesis of xeroderma pigmentosum, and has recently been implicated in Parkinson's disease. The present results will contribute to our understanding of neuronal diseases induced by ultraviolet-induced DNA damage.
- Published
- 2019
46. Degradation of poly aromatic fractions of crude oil and detection of catabolic genes in hydrocarbon-degrading bacteria isolated from Agbabu bitumen sediments in Ondo State
- Author
-
Temitayo O. Olowomofe, Olusola Oluwole, B.I. Aderiye, and J. O. Oluyege
- Subjects
Microbiology (medical) ,lcsh:QR1-502 ,Bacillus cereus ,AlkB ,Dyadobacter koreensis ,medicine.disease_cause ,Microbiology ,biodegradation ,lcsh:Microbiology ,bioremediation ,poly aromatic hydrocarbon ,medicine ,Food science ,crude oil ,chemistry.chemical_classification ,Light crude oil ,biology ,Chemistry ,fungi ,biology.organism_classification ,Hydrocarbon ,Cereus ,gas chromatography/mass spectroscopy ,biology.protein ,bacteria ,Micrococcus luteus ,Bacteria ,Research Article - Abstract
Pollution due to release of Poly aromatic hydrocarbons (PAHs) are a major environmental issue especially in oil producing communities. This study investigates the polyaromatic hydrocarbon degradation potentials of some bacteria: Campylobacter hominis, Bacillus cereus, Dyadobacter koreensis, Pseudomonas aeruginosa and Micrococcus luteus isolated from Agbabu bitumen sediments in Ondo State. The isolates were used singly and in consortium for the degradation of Bonny light crude oil. Concentrations of residual aromatic hydrocarbons in crude oil degraded by these isolates were determined by Gas chromatography/Mass Spectroscopy with flame ionization detector (FID). Detection of catabolic genes (nahH, CatA and AlkB) in the isolates was determined by PCR amplification of their specific primers. The GC-MS analyses showed degradation of poly aromatic hydrocarbons (PAHs) by these isolates. The consortium exhibited the highest PAH reduction (73%) while C. hominis had the least PAH reduction (56%). Dyadobacter koreensis, P. aeruginosa, Micrococcus luteus and B. cereus, displayed 66%, 60%, 59% and 58% PAH reduction respectively. The catabolic gene nahH gene was present in B. cereus, D. koreensis, P. aeruginosa and M. luteus, alkB gene was present in B. cereus, C. hominis, and D. koreensis while CatA was not detected in any of the isolates. The findings of this study affirmed the hydrocarbon-degrading abilities and presence of catabolic genes in these bacteria, these make them potential tools in oil prospecting and cleaning up of hydrocarbon contaminated sites.
- Published
- 2019
47. The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions
- Author
-
Kara A. Bernstein, Hani S. Zaher, Tony M. Mertz, Thong T Luong, Sarah R. Hengel, Braulio Bonilla, Catherine A. Pressimone, Steven A. Roberts, Adeola A Fagunloye, Nima Mosammaparast, Kyle S Rapchak, Alexander J. Brown, Reagan A Russell, Ewa P. Malc, Debra Mitchell, Piotr A. Mieczkowski, and Rudri K Vyas
- Subjects
Saccharomyces cerevisiae Proteins ,Shu complex ,Alkylation ,DNA repair ,QH301-705.5 ,Science ,RAD51 ,AlkB ,Mutagenesis (molecular biology technique) ,S. cerevisiae ,homologous recombination ,Saccharomyces cerevisiae ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Biochemistry and Chemical Biology ,Biology (General) ,alkyation damage ,General Immunology and Microbiology ,biology ,Chemistry ,General Neuroscience ,Rad51 paralogs ,General Medicine ,Cell Biology ,Methyl Methanesulfonate ,Cell biology ,Methyl methanesulfonate ,Mutagenesis ,biology.protein ,Rad51 ,Medicine ,Homologous recombination ,DNA ,Research Article ,Mutagens - Abstract
Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.
- Published
- 2021
48. Computational Investigations of Selected Enzymes From Two Iron and α–ketoglutarate–Dependent Families
- Author
-
G. Andrés Cisneros, Madison B. Berger, Erik A. Vázquez-Montelongo, and Alice R. Walker
- Subjects
chemistry.chemical_classification ,biology ,Escherichia coli Proteins ,Iron ,Mutagenesis ,AlkB Enzymes ,AlkB ,General Physics and Astronomy ,Active site ,RNA ,Molecular Dynamics Simulation ,Article ,DNA Alkylation ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,Transcriptional regulation ,biology.protein ,Escherichia coli ,Ketoglutaric Acids ,Physics - Biological Physics ,Physical and Theoretical Chemistry ,DNA - Abstract
DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.
- Published
- 2021
49. Elucidation of the molecular interactions that enable stable interaction between HIV protease inhibitor ritonavir and human DNA repair enzyme ALKBH2: a molecular dynamics simulation study
- Author
-
Roy Anindya and Monisha Mohan
- Subjects
biology ,Chemistry ,DNA repair ,In silico ,AlkB ,medicine.disease_cause ,DNA Alkylation ,genomic DNA ,biology.protein ,Cancer research ,medicine ,HIV Protease Inhibitor ,Ritonavir ,Carcinogenesis ,medicine.drug - Abstract
The human DNA repair enzyme AlkB homologue-2 and 3 (ALKBH2 and ALKKBH3) repairs methyl adducts from genomic DNA. Overexpression of ALKBH2 and ALKBH3 has been implicated in both tumorigenesis and chemotherapy resistance in some cancers, including glioblastoma and renal cancer rendering it a potential therapeutic target and a diagnostic marker. However, no inhibitor is available against these important DNA repair proteins. Intending to repurpose a drug as an inhibitor of ALKBH2/ALKBH3, we performedin silicoevaluation of HIV protease inhibitors and identified Ritonavir as an ALKBH2-interacting molecule. Using molecular dynamics simulation, we elucidated the molecular details of Ritonavir-ALKBH2 interaction. The present work highlights that Ritonavir might be used to target the ALKBH2-mediated DNA alkylation repair.
- Published
- 2021
50. DNA Demethylation in the Processes of Repair and Epigenetic Regulation Performed by 2-Ketoglutarate-Dependent DNA Dioxygenases
- Author
-
Olga S. Fedorova, Lyubov Yu. Kanazhevskaya, and Nikita A. Kuznetsov
- Subjects
oxygen activation ,DNA Repair ,QH301-705.5 ,AlkB ,Review ,catalytic mechanism ,Catalysis ,Epigenesis, Genetic ,Inorganic Chemistry ,chemistry.chemical_compound ,direct repair ,DNA dioxygenase ,Animals ,Humans ,Epigenetics ,Biology (General) ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,Spectroscopy ,chemistry.chemical_classification ,biology ,epigenetics ,Chemistry ,Organic Chemistry ,AlkB Enzymes ,General Medicine ,Methylation ,DNA Methylation ,Computer Science Applications ,DNA demethylation ,Enzyme ,Biochemistry ,DNA methylation ,Nucleic acid ,biology.protein ,DNA - Abstract
Site-specific DNA methylation plays an important role in epigenetic regulation of gene expression. Chemical methylation of DNA, including the formation of various methylated nitrogenous bases, leads to the formation of genotoxic modifications that impair DNA functions. Despite the fact that different pathways give rise to methyl groups in DNA, the main pathway for their removal is oxidative demethylation, which is catalyzed by nonheme Fe(II)/α-ketoglutarate–dependent DNA dioxygenases. DNA dioxygenases share a common catalytic mechanism of the oxidation of the alkyl groups on nitrogenous bases in nucleic acids. This review presents generalized data on the catalytic mechanism of action of DNA dioxygenases and on the participation of typical representatives of this superfamily, such as prokaryotic enzyme AlkB and eukaryotic enzymes ALKBH1–8 and TET1–3, in both processes of direct repair of alkylated DNA adducts and in the removal of an epigenetic mark (5-methylcytosine).
- Published
- 2021
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