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Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin, and insulin, each of which is stored in electron-dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the ER lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by peptidylglycine α-amidating monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes, and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell types., (© 2021 Federation of European Biochemical Societies.)

Circular RNAs (circRNAs) are key regulators in the development of many cancers. The present study was aimed to investigate the mechanism by which circ_0007919 affected colorectal cancer (CRC) progression.The differentially expressed circRNA was screened out by analyzing the expression profile of circRNAs of CRC tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for detecting the expressions of circ_0007919, miR-942-5p, and ten-eleven translocation 1 (TET1) mRNA in CRC tissues and cell lines. Cell growth and migration were assessed by cell counting kit-8 (CCK-8) 5-bromo-2'-deoxyuridine (BrdU) and scratch assays. Bioinformatics analysis and dual-luciferase reporter assay were conducted to predict and validate the targeted relationships between circ_0007919 and miR-942-5p, as well as between miR-942-5p and TET1 mRNA. Besides, Western blot was conducted for detecting TET1 protein expression in CRC cells. It was revealed that, in CRC tissues and cell lines, circ_0007919 and TET1 expressions were reduced whereas miR-942-5p expression was enhanced. It was also revealed that circ_0007919 overexpression markedly suppressed CRC cell growth and migration. In addition, circ_0007919 could competitively bind with miR-942-5p to increase the expression of miR-942-5p's target gene TET1. Collectively, circ_0007919 inhibits CRC cell growth and migration via regulating the miR-942-5p/TET1 axis. This study helps to better understand the molecular mechanism of CRC progression., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Peptides derived from proopiomelanocortin (POMC) are well-established neuropeptides and peptide hormones that perform multiple functions, including regulation of body weight. In humans and some animals, these peptides include α- and β-melanocyte-stimulating hormone (MSH). In certain rodent species, no β-MSH is produced from POMC because of a change in the cleavage site. Enzymes that convert POMC into MSH include prohormone convertases (PCs), carboxypeptidases (CPs), and peptidyl-α-amidating monooxygenase (PAM). Humans and mice with inactivating mutations in either PC1/3 or carboxypeptidase E (CPE) are obese, which was assumed to result from defective processing of POMC into MSH. However, recent studies have shown that selective loss of either PC1/3 or CPE in POMC-expressing cells does not cause obesity. These findings suggest that defects in POMC processing cannot alone account for the obesity observed in global PC1/3 or CPE mutants. We propose that obesity in animals lacking PC1/3 or CPE activity depends, at least in part, on deficient processing of peptides in non-POMC-expressing cells either in the brain and/or the periphery. Genetic background may also contribute to the manifestation of obesity., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Cellular hypoxia plays a crucial role in tissue development and adaptation to pO 2 . Central to cellular oxygen sensing is factor-inhibiting HIF-1α (FIH), an α-ketoglutarate (αKG)/non-heme iron(II)-dependent dioxygenase that hydroxylates a specific asparagine residue of hypoxia inducible factor-1α (HIF-1α). The high K M(O 2 ) and rate-limiting decarboxylation step upon O 2 activation are key features of the enzyme that classify it as an oxygen sensor and set it apart from other αKG/Fe(II)-dependent dioxygenases. Although the chemical intermediates following decarboxylation are presumed to follow the consensus mechanism of other αKG/Fe(II)-dependent dioxygenases, experiments have not previously demonstrated these canonical steps in FIH. In this work, a deuterated peptide substrate was used as a mechanistic probe for the canonical hydrogen atom transfer (HAT). Our data show a large kinetic isotope effect (KIE) in steady-state kinetics ( D k cat = 10 ± 1), revealing that the HAT occurs and is partially rate limiting on k cat . Kinetic studies showed that the deuterated peptide led FIH to uncouple O 2 activation and provided the opportunity to spectroscopically observe the ferryl intermediate. This enzyme uncoupling was used as an internal competition with respect to the fate of the ferryl intermediate, demonstrating a large observed KIE on the uncoupling ( D k 5 = 1.147 ± 0.005) and an intrinsic KIE on the HAT step ( D k > 15). The close energy barrier between αKG decarboxylation and HAT distinguishes FIH as an O 2 -sensing enzyme and is crucial for ensuring substrate specificity in the regulation of cellular O 2 homeostasis.

MiR-143-3p is aberrantly expressed in patients with ischemic stroke and associated with ischemic brain injury. However, the underlying mechanisms are largely unknown. Here, we confirmed circ_0025984 and TET1 as a sponge and target of miR-143-3p, respectively, by luciferase reporter assay. In astrocytes, OGD significantly decreased circ_0025984 and TET1 levels but increased miR-143-3p levels, which was also observed in brains of mice with MCAO. Treatment with miR-143-3p inhibitor or circ_0025984 significantly decreased astrocyte apoptosis and autophagy, as well as cerebral injury and neuron loss in mice with MCAO. Notably, TET1 overexpression decreased astrocyte apoptosis and autophagy and induced promoter hypomethylation and expression of ORP150. Our results demonstrated for the first time that circ_0025984 protects astrocytes from ischemia-induced autophagy and apoptosis by targeting the miR-143-3p/TET1 pathway and might inhibit cerebral injury induced by ischemic stroke. Furthermore, our data revealed the important positive regulation of ORP150 by TET1, which could be associated with its neuroprotective role. Graphical abstract Model for signaling pathway of circ_0025984/miR-143-3p/TET1 inastrocytes cultured under OGD. In astrocytes, circ_0025984 acts as a sponge of miR-143-3p, which directly targets TET1 and decreases its expression (A). After translocatinginto the nucleus, TET1 binds to the promoter of ORP150, converts 5mC into 5hmC,leading to DNA demethylation and increased expression of ORP150 (B). In astrocytescultured under OGD, ER stress is induced and eventually leads to apoptosis andautophagy mediated by ATG7, which is regulated by circ_0025984 via ORP150 andGRP78 (C)., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Gibberellin 2-oxidase (GA2ox) plays an important role in the GA catabolic pathway and the molecular function of the OsGA2ox genes in plant abiotic stress tolerance remains largely unknown. In this study, we functionally characterized the rice gibberellin 2-oxidase 8 ( OsGA2ox8 ) gene. The OsGA2ox8 protein was localized in the nucleus, cell membrane, and cytoplasm, and was induced in response to various abiotic stresses and phytohormones. The overexpression of OsGA2ox8 significantly enhanced the osmotic stress tolerance of transgenic rice plants by increasing the number of osmotic regulators and antioxidants. OsGA2ox8 was differentially expressed in the shoots and roots to cope with osmotic stress. The plants overexpressing OsGA2ox8 showed reduced lengths of shoots and roots at the seedling stage, but no difference in plant height at the heading stage was observed, which may be due to the interaction of OsGA2ox8 and OsGA20ox1, implying a complex feedback regulation between GA biosynthesis and metabolism in rice. Importantly, OsGA2ox8 was able to indirectly regulate several genes associated with the anthocyanin and flavonoid biosynthetic pathway and the jasmonic acid (JA) and abscisic acid (ABA) biosynthetic pathway, and overexpression of OsGA2ox8 activated JA signal transduction by inhibiting the expression of jasmonate ZIM domain-containing proteins. These results provide a basis for a future understanding of the networks and respective phenotypic effects associated with OsGA2ox8 .

C-terminal α-amidation is the final and essential step in the biosynthesis of several peptide hormones. Peptidylglycine α-amidating monooxygenase (PAM) is the only known enzyme to catalyse this reaction. PAM amidating activity (AMA) is known to be present in human circulation, but its physiological role and significance as a clinical biomarker remains unclear. We developed a PAM-specific amidation assay that utilizes the naturally occurring substrate Adrenomedullin-Gly (ADM-Gly, 1-53). Using our amidation assay we quantified serum amidating activities in a large population-based cohort of more than 4900 individuals. A correlation of serum amidating activity with several clinical parameters including high blood pressure was observed. Increasing PAM-AMA was an independent predictor of hard outcomes related to hemodynamic stress such as cardiovascular mortality, atrial fibrillation and heart failure during long-term follow-up (8.8 ± 2.5 years). Moreover, results from an animal study in rats utilizing recombinant human PAM provide novel insights into the physiological role of circulating PAM and show its potential significance in circulating peptide amidation., (© 2021. The Author(s).)

Hepatocellular carcinoma (HCC), one of the main contributors to cancer-associated deaths globally, is characterized by high invasiveness. Herein, we studied the molecular mechanisms underlying ten-eleven translocation 1 (TET1)-mediated autophagy in HCC. Following data mining using GSE101728, GSE14520 and GSE138178, TET1 was screened out, and the differential expression of TET1 was verified by bioinformatics analysis. TET1, one of the prognostic markers in HCC, was poorly expressed in HCC. Through functional experiments, we determined that upregulation of TET1 inhibited the proliferation, migration, invasion, tumorigenesis, metastasis and inflammatory factors of HCC cells, and promoted cell autophagy and apoptosis. Mechanistically, TET1 activated miR-34a by demethylating miR-34a. BTB domain and CNC homology 1 (BACH1) was identified as the target gene of miR-34a. Notably, Downregulation of miR-34a increased cellular inflammatory factors and decreased autophagy in the presence of TET1, while declines in BACH1 suppressed cellular inflammatory factors and enhanced autophagy in the presence of miR-34a inhibitor. BACH1 negatively regulated the p53 pathway. In conclusion, TET1 is a tumor suppressor in the progression of HCC by regulating the miR-34a/BACH1/p53 axis, and may contribute to the improvement of HCC prognosis and therapy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes., (© 2021 The Author(s).)

Abscisic acid (ABA) is a key hormone in non-climacteric Fragaria spp, regulating multiple physiological processes throughout fruit ripening. Its concentration increases during ripening, and it promotes fruit (receptacle) development. However, its metabolism in the fruit is largely unknown. We analyzed the concentrations of ABA and its catabolites at different developmental stages of strawberry ripening in diploid and octoploid genotypes and identified two functional ABA-glucosyltransferases (FvUGT71A49 and FvUGT73AC3) and two regiospecific ABA-8'-hydroxylases (FaCYP707A4a and FaCYP707A1/3). ABA-glucose ester content increased during ripening in diploid F. vesca varieties but decreased in octoploid F.×ananassa. Dihydrophaseic acid content increased throughout ripening in all analyzed receptacles, while 7'-hydroxy-ABA and neo-phaseic acid did not show significant changes during ripening. In the studied F. vesca varieties, the receptacle seems to be the main tissue for ABA metabolism, as the concentration of ABA and its metabolites in the receptacle was generally 100 times higher than in achenes. The accumulation patterns of ABA catabolites and transcriptomic data from the literature show that all strawberry fruits produce and metabolize considerable amounts of the plant hormone ABA during ripening, which is therefore a conserved process, but also illustrate the diversity of this metabolic pathway which is species, variety, and tissue dependent., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Processing of and responding to various signals is an essential cellular function that influences survival, homeostasis, development, and cell death. Extra- or intracellular signals are perceived via specific receptors and transduced in a particular signalling pathway that results in a precise response. Reversible post-translational redox modifications of cysteinyl and methionyl residues have been characterised in countless signal transduction pathways. Due to the low reactivity of most sulfur-containing amino acid side chains with hydrogen peroxide, for instance, and also to ensure specificity, redox signalling requires catalysis, just like phosphorylation signalling requires kinases and phosphatases. While reducing enzymes of both cysteinyl- and methionyl-derivates have been characterised in great detail before, the discovery and characterisation of MICAL proteins evinced the first examples of specific oxidases in signal transduction. This article provides an overview of the functions of MICAL proteins in the redox regulation of cellular functions., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aims/hypothesis: Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes., Methods: PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam +/- and Pam +/+ mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam +/- and Pam +/+ mice with beta cell expression of hIAPP., Results: Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1β, IFN-γ and TFN-α. Despite Pam +/- only having 50% PAM expression and enzyme activity as compared with Pam +/+ mice, glucose tolerance and body mass composition were comparable in the two models. After 24 weeks of HFD, both Pam +/- and Pam +/+ mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam +/- and Pam +/+ mice with beta cell expression of hIAPP., Conclusions/interpretation: Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

Congenital heart disease (CHD) is a major cause of morbidity in the pediatric population yet its genetic and molecular causes remain poorly defined. Previously, we identified AGMO as a candidate heterotaxy disease gene, a disorder of left-right (LR) patterning that can have a profound effect on cardiac function. AGMO is the only known alkylglycerol monooxygenase, an orphan tetrahydrobiopterin dependent enzyme that cleaves the ether linkage in alkylglycerols. However, whether AGMO plays a role in LR patterning was unexplored. Here we reveal that Agmo is required for correct development of the embryonic LR axis in Xenopus embryos recapitulating the patient's heterotaxy phenotype. Mechanistically, we demonstrate that Agmo is a regulator of canonical Wnt signaling, required during gastrulation for normal formation of the left - right organizer. Mutational analysis demonstrates that this function is dependent on Agmo's alkylglycerol monooxygenase activity. Together, our findings identify Agmo as a regulator of canonical Wnt signaling, demonstrate a role for Agmo in embryonic axis formation, and provide insight into the poorly understood developmental requirements for ether lipid cleavage., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The dynamic nature of epigenetic DNA modifications is crucial for regulating gene expression in an experience-dependent manner and, thus, a potential mediator of neuronal plasticity and behavior. The discovery of the involvement of 5-hydroxymethylcytosine (5hmC) and Ten Eleven Translocation (TET) family of enzymes in the demethylation pathway uncovered a potential link between neuronal TET protein function and cognitive processes. In this review, we provide an overview on how profile of 5hmC and TET enzymes are powerful mechanisms to explain neuronal plasticity and long-term behaviors, such as cognition. More specifically, we discuss how the current knowledge integrates the function of each TET enzyme in neurophysiology and brain function., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

All metazoans that utilize molecular oxygen (O 2 ) for metabolic purposes have the capacity to adapt to hypoxia, the condition that arises when O 2 demand exceeds supply. This is mediated through activation of the hypoxia-inducible factor (HIF) pathway. At physiological oxygen levels (normoxia), HIF-prolyl hydroxylases (PHDs) hydroxylate proline residues on HIF-α subunits leading to their destabilization by promoting ubiquitination by the von-Hippel Lindau (VHL) ubiquitin ligase and subsequent proteasomal degradation. HIF-α transactivation is also repressed in an O 2 -dependent way due to asparaginyl hydroxylation by the factor-inhibiting HIF (FIH). In hypoxia, the O 2 -dependent hydroxylation of HIF-α subunits by PHDs and FIH is reduced, resulting in HIF-α accumulation, dimerization with HIF-β and migration into the nucleus to induce an adaptive transcriptional response. Although HIFs are the canonical substrates for PHD- and FIH-mediated protein hydroxylation, increasing evidence indicates that these hydroxylases may also have alternative targets. In addition to PHD-conferred alterations in protein stability, there is now evidence that hydroxylation can affect protein activity and protein/protein interactions for alternative substrates. PHDs can be pharmacologically inhibited by a new class of drugs termed prolyl hydroxylase inhibitors which have recently been approved for the treatment of anemia associated with chronic kidney disease. The identification of alternative targets of HIF hydroxylases is important in order to fully elucidate the pharmacology of hydroxylase inhibitors (PHI). Despite significant technical advances, screening, detection and verification of alternative functional targets for PHDs and FIH remain challenging. In this review, we discuss recently proposed non-HIF targets for PHDs and FIH and provide an overview of the techniques used to identify these.

The gene lpmo1 encoding Penicillium verruculosum lytic polysaccharide monooxygenase (PvLPMO9A) was sequenced and homologously overexpressed in P. verruculosum B1-537 (ΔniaD) auxotrophic strain under the control of the cbh1 gene promoter in combination with either the cbh1 signal sequence (sCBH1-X series of samples) or the native lpmo1 signal sequence (sLPMO1-X series). Three enzyme samples of the sCBH1-X series were characterized by a lower overall content of cellobiohydrolases (CBHs: 26-45%) but slightly higher content of endoglucanases (EGs: 17-23%) relative to the reference B1-537 preparation (60% of CBHs and 14% of EGs), while the PvLPMO9A content in them made up 9-21% of the total secreted protein. The PvLPMO9A content in four enzyme preparations of the sLPMO1-X series was much higher (30-57%), however the portion of CBHs in most of them (except for sLPMO1-8) decreased even to a greater extent (to 21-42%) than in the samples of the sCBH1-X series. Two enzyme preparations (sCBH1-8 and sLPMO1-8), in which the content of cellulases was substantially retained and the portion of PvLPMO9A was 9-30%, demonstrated the increased yields of reducing sugars in 48-h saccharification of Avicel and milled aspen wood: 19-31 and 11-26%, respectively, compared to the reference cellulase cocktail.

In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B. megaterium system, yielding about 45 mg L -1 of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Some carotenoids are found in the Euglena gracilis, including β-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated β-carotene to β-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of β-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be β-carotene monohydroxylase, but not β-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Caleosin/peroxygenases, CLO/PXG, (designated PF05042 in Pfam) are a group of genes/proteins with anomalous distributions in eukaryotic taxa. We have previously characterised CLO/PXGs in the Viridiplantae. The aim of this study was to investigate the evolution and functions of the CLO/PXGs in the Fungi and other non-plant clades and to elucidate the overall origin of this gene family., Results: CLO/PXG-like genes are distributed across the full range of fungal groups from the basal clades, Cryptomycota and Microsporidia, to the largest and most complex Dikarya species. However, the genes were only present in 243 out of 844 analysed fungal genomes. CLO/PXG-like genes have been retained in many pathogenic or parasitic fungi that have undergone considerable genomic and structural simplification, indicating that they have important functions in these species. Structural and functional analyses demonstrate that CLO/PXGs are multifunctional proteins closely related to similar proteins found in all major taxa of the Chlorophyte Division of the Viridiplantae. Transcriptome and physiological data show that fungal CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression and are upregulated in response to a range of biotic and abiotic stresses as well as participating in key metabolic and developmental processes such as lipid metabolism, signalling, reproduction and pathogenesis. Biochemical data also reveal that the Aspergillus flavus CLO/PXG has specific functions in sporulation and aflatoxin production as well as playing roles in lipid droplet function., Conclusions: In contrast to plants, CLO/PXGs only occur in about 30% of sequenced fungal genomes but are present in all major taxa. Fungal CLO/PXGs have similar but not identical roles to those in plants, including stress-related oxylipin signalling, lipid metabolism, reproduction and pathogenesis. While the presence of CLO/PXG orthologs in all plant genomes sequenced to date would suggest that they have core housekeeping functions in plants, the selective loss of CLO/PXGs in many fungal genomes suggests more restricted functions in fungi as accessory genes useful in particular environments or niches. We suggest an ancient origin of CLO/PXG-like genes in the 'last eukaryotic common ancestor' (LECA) and their subsequent loss in ancestors of the Metazoa, after the latter had diverged from the ancestral fungal lineage.

Key Points: Gestational hypoxia represses ten-eleven translocation methylcytosine dioxygenase 1 (TET1) expression in uterine arteries, which is recovered by inhibiting endogenous miR-210. Inhibition of miR-210 rescues BK Ca channel expression and current in uterine arteries of pregnant animals acclimatized to high altitude hypoxia in a TET-dependent manner. miR-210 blockade restores BK Ca channel-mediated relaxations and attenuates pressure-dependent myogenic tone in uterine arteries of pregnant animals acclimatized to high altitude., Abstract: Gestational hypoxia at high altitude has profound adverse effects on the uteroplacental circulation, and is associated with increased incidence of preeclampsia and fetal intrauterine growth restriction. Previous studies demonstrated that suppression of large-conductance Ca 2+ -activated K + (BK Ca ) channel function played a critical role in the maladaptation of uteroplacental circulation caused by gestational hypoxia. Yet, the mechanisms underlying gestational hypoxia-induced BK Ca channel repression remain undetermined. The present study investigated a causal role of microRNA-210 (miR-210) in hypoxia-mediated repression of BK Ca channel expression and function in uterine arteries using a sheep model. The results revealed that gestational hypoxia significantly decreased ten-eleven translocation methylcytosine dioxygenase 1 (TET1) expression in uterine arteries, which was recovered by inhibiting endogenous miR-210 with miR-210 locked nucleic acid (miR-210-LNA). Of importance, miR-210-LNA restored BK Ca channel β1 subunit expression in uterine arteries, which was blocked by a competitive TET inhibitor, fumarate, thus functionally linking miR-210 to the TET1-BK Ca channel cascade. In addition, miR-210-LNA reversed hypoxia-mediated suppression of BK Ca channel function and rescued the effect of steroid hormones in upregulating BK Ca channel expression and function in uterine arteries, which were also ablated by fumarate. Collectively, the present study demonstrates a causative effect of miR-210 in the downregulation of TET1 and subsequent repression of BK Ca channel expression and function, providing a novel mechanistic insight into the regulation of BK Ca channel function and the molecular basis underlying the maladaptation of uterine vascular function in gestational hypoxia., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Plants substantially alter their developmental programme upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15°C, 21°C, and 27°C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive microRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature-dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants., (© 2018 John Wiley & Sons Ltd.)

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis., Competing Interests: The authors have declared that no competing interests exists.

Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in yuc2yuc6 double mutants arrests before PMI and consequently yuc2yuc6 fail to produce viable pollens. Our genetic analyses reveal that YUC2 and YUC6 act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of yuc2yuc6. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in yuc2yuc6 double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte., Competing Interests: The authors have declared that no competing interests exist.

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Methylation at cytosine (5mC) is a fundamental epigenetic DNA modification recently associated with iAs-mediated carcinogenesis. In contrast, the role of 5-hydroxymethylcytosine (5hmC), the oxidation product of 5mC in iAs-mediated carcinogenesis is unknown. Here we assess the hydroxymethylome in iAs-transformed cells, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks. Moreover, this pathologic iAs-mediated carcinogenesis is characterized by a shift toward a higher hydroxymethylation pattern genome-wide. At specific promoters, hydroxymethylation correlated with increased gene expression. Furthermore, this increase in hydroxymethylation occurs concurrently with an upregulation of ten-eleven translocation (TET) enzymes that oxidize 5-methylcytosine (5mC) in DNA. To gain an understanding into how iAs might impact TET expression, we found that iAs inhibits the binding of CTCF at the proximal, weak CTCF binding sites of the TET1 and TET2 gene promoters and enhances CTCF binding at the stronger distal binding site. Further analyses suggest that this distal site acts as an enhancer, thus high CTCF occupancy at the enhancer region of TET1 and TET2 possibly drives their high expression in iAs-transformed cells. These results have major implications in understanding the impact of differential CTCF binding, genome architecture and its consequences in iAs-mediated pathogenesis., (Copyright © 2017. Published by Elsevier Inc.)

The development of the nervous system is coordinately regulated by multiple interacting factors. If a certain factor is altered or mutated, the coordinated developmental processes could be disrupted, resulting in neurological diseases. The 5-hydroxymethylcytosine (5hmC) is an intermediate product of the DNA demethylation processes. 5hmC and its metabolic enzymes, the ten-eleven translocation protein-TET family of dioxygenases, have recently been identified as new epigenetic players important in the regulation of the nervous system development, as well as in cognition, memory and other neurological functions. In various studies on neurodevelopment and neurodegeneration related diseases, the levels of 5hmC and TET proteins could be differentially regulated during development and/or disease pathogenesis, suggesting the potentially critical roles of 5hmC and TETs in these neural developmental and disease processes. In this review, we summarize the recent advances in research on 5hmC and TET dioxygenases in the regulation of neurodevelopment and neurological diseases, thereby providing significant insights on the involvements of 5hmC and TETs in neurodevelopment and on establishing new therapeutic strategies for human neurological diseases.

The development of chemo-resistance impairs the outcome of the first line platinum-based chemotherapies for ovarian cancer. Deregulation of DNA methylation/demethylation provides a critical mechanism for the occurrence of chemo-resistance. The ten-eleven translocation (TET) family of dioxygenases including TET1/2/3 plays an important part in DNA demethylation, but their roles in cisplatin resistance have not been elucidated. Using cisplatin-sensitive and cisplatin-resistant ovarian cancer cell models, we found that TET1 was significantly upregulated in cisplatin-resistant CP70 cells compared with that in cisplatin-sensitive A2780 cells. Ectopic expression of TET1 in A2780 cells promoted cisplatin resistance and decreased cytotoxicity induced by cisplatin, while inhibition of TET1 by siRNA transfection in CP70 cells attenuated cisplatin resistance and enhanced cytotoxicity of cisplatin. Increased TET1 induced re-expression of vimentin through active DNA demethylation, and cause partial epithelial-to-mesenchymal (EMT) in A2780 cells. Contrarily, knocking down of TET1 in CP70 cells reduced vimentin expression and reversed EMT process. Immunohistochemical analysis of TET1 in human ovarian cancer tissues revealed that TET1 existed in nucleus and cytoplasm in ovarian cancer tissues. And the expression of nuclear TET1 was positively correlated with residual tumor and chemotherapeutic response. Thus, TET1 expression causes resistance to cisplatin and one of the targets of TET1 action is vimentin in ovarian cancer., (© 2017 International Federation for Cell Biology.)

Chloroplasts are essential for photosynthesis and play critical roles in plant development. In this study, we characterized the temperature-sensitive chlorophyll-deficient rice mutant tcd5, which develops albino leaves at low temperatures (20 °C) and normal green leaves at high temperatures (32 °C). The development of chloroplasts and etioplasts is impaired in tcd5 plants at 20 °C, and the temperature-sensitive period for the albino phenotype is the P4 stage of leaf development. The development of thylakoid membranes is arrested at the mid-P4 stage in tcd5 plants at 20 °C. We performed positional cloning of TCD5 and then complementation and knock-down experiments, and the results showed that the transcript LOC_Os05g34040.1 from the LOC_Os05g34040 gene corresponded to the tcd5 phenotype. TCD5 encodes a conserved plastid-targeted monooxygenase family protein which has not been previously reported associated with a temperature-sensitive albino phenotype in plants. TCD5 is abundantly expressed in young leaves and immature spikes, and low temperatures increased this expression. The transcription of some genes involved in plastid transcription/translation and photosynthesis varied in the tcd5 mutant. Although the phenotype and temperature dependence of the TCD5 orthologous mutant phenotype were different in rice and Arabidopsis, OsTCD5 could rescue the phenotype of the Arabidopsis mutant, suggesting that TCD5 function is conserved between monocots and dicots., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.

The hepatoprotectant flavonolignan silymarin (Sm) is synthesized through 4-coumaroyl-CoA, which enters both the flavonoid and the monolignol pathway giving the two immediate precursors taxifolin (Tx) and coniferyl alcohol (CA), respectively. Sm formation occurs via oxidative radicalization of Tx and CA and is accumulated at high levels at final stages of maturation of Silybum marianum fruits. By contrast, Sm production is severely reduced in cell cultures of this species, although suspensions are able to excrete Sm compounds into the medium upon elicitation with methyl jasmonate (MeJA) or cyclodextrins (CD). Knowledge of gene expression is important to understand Sm dynamics and to develop strategies aimed at increasing production by means of cell cultures but, to date, only one gene of the pathway (chalcone synthase, SmCHS) has been cloned. Therefore, to elucidate the relationship between expression of Sm pathway genes and production of these metabolites, four cDNA fragments of genes putatively involved in flavonolignan biosynthesis, chalcone isomerase, flavanone 3-hydroxylase, flavonol 3'-hydroxylase and cinnamyl alcohol dehydrogenase, were isolated from Sm producing S. marianum fruits and their expression, together with that of the SmCHS, were studied both in fruits at different maturation stages and in elicited cell suspensions. Combined results at both transcript expression and metabolite levels at three different stages of fruit maturation revealed that the formation of the flavonoid moiety precedes flavonolignan biosynthesis, being Sm accumulation associated to expression of the monolignol pathway. There was not detectable accumulation of transcripts in cell suspensions, however, elicitation with MeJA or CD notably induced expression of the studied fragments. These results indicate that the five genes expressed during maturation of S. marianum fruits may contribute to observed increases in flavonolignan accumulation upon treatment of cell cultures with elicitors., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

We studied the role of the liver monooxygenase system in pharmacological activity of histochrome, a pharmaceutical form of echinochrome A. In experiments on rats, benzonal, an inductor of the monooxygenase system of phenobarbytal type, significantly potentiated the diuretic effect of histochrome. Benzonal withdrawal was followed by a natriuretic reaction. In view of the known inverse relation between the biological effect of the drug and the rate of its metabolism, our findings suggest that the effects of echinochrome A on some kidney excretory function parameters are produced not by native agent, but one of its metabolites.

Nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease among children and adolescents in the developed world. Betaine, as a methyl donor, recently has been demonstrated to exert its hepatoprotective effects through rectifying the genomic DNA hypomethylation state. However, whether betaine supplementation affects N6-methyladenosine (m(6)A) mRNA methylation in NAFLD is still unknown. We conducted the current study to investigate the effects of betaine supplementation during adolescence on high-fat diet-induced pathological changes in liver of mice, and we further identified the effects of betaine supplementation on expression of the fat mass and obesity-associated gene (FTO) and hepatic m(6)A mRNA methylation. Our results showed that betaine supplementation across adolescence significantly alleviated high-fat-induced impairment of liver function and morphology as well as ectopic fat accumulation. Surprisingly, no significant effects on serum TG and NEFA level, as well as fat mass, were observed in mice supplemented with betaine. We also found that high-fat diet upregulated ACC1 and FAS gene expression and downregulated HSL and ATGL gene expression. However, these alterations were rectified by betaine supplementation. Moreover, an m(6)A hypomethylation state and increased FTO expression were detected in mice fed with high-fat diet, while betaine supplementation prevented these changes. Our results suggested that betaine supplementation during adolescence could protect mice from high-fat-induced NAFLD by decreasing de novo lipogenesis and increasing lipolysis. Furthermore, a novel FTO-dependent function of m(6)A may involve in the hepatoprotective effects of betaine.

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hypoxia inducible factors (HIFs) are α/β heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease., (© 2015 American Heart Association, Inc.)

Hydroxylation is an emerging modification generally catalyzed by a family of ∼70 enzymes that are dependent on oxygen, Fe(II), ascorbate, and the Kreb's cycle intermediate 2-oxoglutarate (2OG). These "2OG oxygenases" sit at the intersection of nutrient availability and metabolism where they have the potential to regulate gene expression and growth in response to changes in co-factor abundance. Characterized 2OG oxygenases regulate fundamental cellular processes by catalyzing the hydroxylation or demethylation (via hydroxylation) of DNA, RNA, or protein. As such they have been implicated in various syndromes and diseases, but particularly cancer. In this review we discuss the emerging role of 2OG oxygenases in gene expression control, examine the regulation of these unique enzymes by nutrient availability and metabolic intermediates, and describe these properties in relation to the expanding role of these enzymes in cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Global warming is predicted to profoundly affect plant distribution and crop yield in the near future. Higher ambient temperature can influence diverse aspects of plant growth and development. In Arabidopsis, the basic helix-loop-helix transcription factor Phytochrome-Interacting Factor 4 (PIF4) regulates temperature-induced adaptive responses by modulating auxin biosynthesis. At high temperature, PIF4 directly activates expression of YUCCA8 (YUC8), a gene encoding an auxin biosynthetic enzyme, resulting in auxin accumulation. Here we demonstrate that the RNA-binding protein FCA attenuates PIF4 activity by inducing its dissociation from the YUC8 promoter at high temperature. At 28 °C, auxin content is elevated in FCA-deficient mutants that exhibit elongated stems but reduced in FCA-overexpressing plants that exhibit reduced stem growth. We propose that the FCA-mediated regulation of YUC8 expression tunes down PIF4-induced architectural changes to achieve thermal adaptation of stem growth at high ambient temperature.

Introduction: Human dental pulp cells (hDPCs) can specifically generate reparative dentin under external stimuli, and numerous mechanisms are involved in their odontogenic differentiation process. Ten-eleven translocation 1 (TET1) is a recently discovered DNA dioxygenase that plays important roles in promoting DNA demethylation and transcriptional regulation. Although several studies regarding its effect on cell differentiation and proliferation have been conducted, the expression and function of TET1 have not yet been characterized in hDPCs. The purpose of this study was to explore the expression features of TET1 in hDPCs., Methods: Cellular TET1 localization in hDPCs was determined by immunofluorescence. The expression pattern of TET1 and its potential changes during odontogenic induction were confirmed using real-time quantitative polymerase chain reaction and Western blot analyses., Results: TET1 existed in both the cytoplasm and the nucleus of the hDPCs. During serial cell passaging, TET1 expression significantly increased until the 6th passage and then decreased from the 7th-9th passages (P < .05, n = 3). TET1 gene and protein expression increased during the odontogenic differentiation of the hDPCs in a time-dependent manner (P < .05, n = 3)., Conclusions: TET1 messenger RNA and protein were both present in the hDPCs. TET1 expression increased during early spontaneous differentiation and odontogenic induction., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The MOXD2 gene encodes a membrane-bound monooxygenase similar to dopamine-β-hydroxylase, and has been proposed to be associated with olfaction. In this study, we analyzed MOXD2 genes from 64 mammalian species, and identified loss-of-function mutations in apes (humans, Sumatran and Bornean orangutans, and five gibbon species from the four major gibbon genera), toothed whales (killer whales, bottlenose dolphins, finless porpoises, baijis, and sperm whales), and baleen whales (minke whales and fin whales). We also identified a shared 13-nt deletion in the last exon of Old World cercopithecine monkeys that results in conversion of a membrane-bound protein to a soluble form. We hypothesize that the frequent inactivation and alteration of MOXD2 genes in catarrhines and whales may be associated with the evolution of olfaction in these clades.

Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

Mical is a reduction-oxidation (redox) enzyme that functions as an unusual F-actin disassembly factor during Drosophila development. Although three Molecule interacting with CasL (MICAL) proteins exist in vertebrate species, their mechanism of action remains poorly defined and their role in vivo unknown. Here, we report that vertebrate MICAL-1 regulates the targeting of secretory vesicles containing immunoglobulin superfamily cell adhesion molecules (IgCAMs) to the neuronal growth cone membrane through its ability to control the actin cytoskeleton using redox chemistry, thereby maintaining appropriate IgCAM cell surface levels. This precise regulation of IgCAMs by MICAL-1 is essential for the lamina-specific targeting of mossy fibre axons onto CA3 pyramidal neurons in the developing mouse hippocampus in vivo. These findings reveal the first in vivo role for a vertebrate MICAL protein, expand the repertoire of cellular functions controlled through MICAL-mediated effects on the cytoskeleton, and provide insights into the poorly characterized mechanisms underlying neuronal protein cell surface expression and lamina-specific axonal targeting.

In mammals, two carotenoid cleaving oxygenases are known; beta-carotene 15,15'-monooxygenase (BCMO1) and beta-carotene 9',10'-oxygenase (BCO2). BCMO1 is a key enzyme in vitamin A synthesis by symmetrically cleaving beta-carotene into 2 molecules of all-trans-retinal, while BCO2 is responsible for asymmetric cleavage of a broader range of carotenoids. Here, we show that the Atlantic salmon beta-carotene oxygenase (bco) gene family contains 5 members, three bco2 and two bcmo1 paralogs. Using public sequence databases, multiple bco genes were also found in several additional teleost species. Phylogenetic analysis indicates that bco2a and bco2b originate from the teleost fish specific genome duplication (FSGD or 3R), while the third and more distant paralog, bco2 like, might stem from a prior duplication event in the teleost lineage. The two bcmo1 paralogs (bcmo1 and bcmo1 like) appear to be the result of an ancient duplication event that took place before the divergence of ray-finned (Actinopterygii) and lobe-finned fish (Sarcopterygii), with subsequent nonfunctionalization and loss of one Sarcopterygii paralog. Gene expression analysis of the bcmo1 and bco2 paralogs in Atlantic salmon reveals regulatory divergence with tissue specific expression profiles, suggesting that the beta-carotene oxygenase subtypes have evolved functional divergences. We suggest that teleost fish have evolved and maintained an extended repertoire of beta-carotene oxygenases compared to the investigated Sarcopterygii species, and hypothesize that the main driver behind this functional divergence is the exposure to a diverse set of carotenoids in the aquatic environment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes - DOXA, DOXB and DOXC - based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4-hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC-class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm-specific clades were found to be involved in various lineage-specific specialized metabolisms, but 31 of the 57 DOXC-class clades were only found in a single species. Therefore, the evolution and diversification of DOXC-class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.

Background and Aim: Alcohol-related liver disease (ALD) is mediated in part by insulin resistance. Attendant dysregulation of lipid metabolism increases accumulation of hepatic ceramides that worsen insulin resistance and compromise the structural and functional integrity of the liver. Insulin and insulin growth factor (IGF) stimulate aspartyl-asparaginyl-β-hydroxylase (AAH), which promotes cell motility needed for structural maintenance and remodeling of the liver. AAH mediates its effects by activating Notch, and in ALD, insulin/IGF signaling, AAH, and Notch are inhibited., Method: To test the hypothesis that in ALD, hepatic ceramide load contributes to impairments in insulin, AAH, and Notch signaling, control and chronic ethanol-fed adult Long-Evans rats were treated with myriocin, an inhibitor of serine palmitoyl transferase. Livers were used to assess steatohepatitis, insulin/IGF pathway activation, and expression of AAH-Notch signaling molecules., Results: Chronic ethanol-fed rats had steatohepatitis with increased ceramide levels; impairments in signaling through the insulin receptor, insulin receptor substrate, and Akt; and decreased expression of AAH, Notch, Jagged, Hairy-Enhancer of Split-1, hypoxia-inducible factor 1α, and proliferating cell nuclear antigen. Myriocin abrogated many of these adverse effects of ethanol, particularly hepatic ceramide accumulation, steatohepatitis, and impairments of insulin signaling through Akt, AAH, and Notch., Conclusions: In ALD, the histopathology and impairments in insulin/IGF responsiveness can be substantially resolved by ceramide inhibitor treatments, even in the context of continued chronic ethanol exposure., (© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Dopaminergic (DA) signaling governs the control of complex behaviors, and its deregulation has been implicated in a wide range of diseases. Here we demonstrate that inactivation of the Fto gene, encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively, 'D2-like receptor')-dependent control of neuronal activity and behavioral responses. Conventional and DA neuron-specific Fto knockout mice show attenuated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channel conductance by cocaine and quinpirole. Impaired D2-like receptor-mediated autoinhibition results in attenuated quinpirole-mediated reduction of locomotion and an enhanced sensitivity to the locomotor- and reward-stimulatory actions of cocaine. Analysis of global N(6)-methyladenosine (m(6)A) modification of mRNAs using methylated RNA immunoprecipitation coupled with next-generation sequencing in the midbrain and striatum of Fto-deficient mice revealed increased adenosine methylation in a subset of mRNAs important for neuronal signaling, including many in the DA signaling pathway. Several proteins encoded by these mRNAs had altered expression levels. Collectively, FTO regulates the demethylation of specific mRNAs in vivo, and this activity relates to the control of DA transmission.

Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D3-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N(4)-ethenocytosine, 1,N(6)-ethenoadenine, 3,N(4)-α-hydroxyethanocytosine, and reported here for the first time 3,N(4)-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK(a) values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N(4)-α-hydroxyethanocytosine or 3,N(4)-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.