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Lichens are composed of symbiotic slow-growing organisms and are often exposed to extreme microenvironmental conditions, resulting in the production of unique secondary metabolites. One of the most commonly produced secondary metabolites is usnic acid, which is thought to be produced by two genes. Theobjectives ofthe present study were to comparepolyketidesynthase(PKS)gene expression and usnic acid concentration in Cladonia uncialis (L.) Weber ex F.H. Wigg., with two environmental factors. Seventy-five lichen samples were collected from three locations in Newfoundland, Canada, using a strip transect method (X5transects, X5 quadrats). Usnic acid concentration was measured using the liquid chromatography tandem mass-spectrometric method and gene expression of two PKS genes (methylphlora-cetophenone oxidase (MPAO) and methylphloracetophenone synthase (MPAS)) was examined using quantitative real-time PCR. The results showed that percent ground cover of C. uncialis was affected by soil pH level but not soil moisture, and usnic acid concentration was not affected by either soil pH or soil moisture. MPAO gene expression level was significantly affected by soil pH level but not soil moisture, and MPAS gene expression level was not affected by either soil pH level or soil moisture. There was no significant relationship between MPAS and MPAO gene expression levels and usnic acid concentration. These findings suggest that soil pH may be important for the production of usnic acid by C. uncialis but the genes involved require further study. Key words: lichen diversity, MPAO, MPAS, percent cover, secondary metabolites, soil moisture content, soil pH. Les lichens sont composés d'organismes symbiotiques à croissance lente et sont souvent exposés à des conditions micro-environnementales extrêmes, ce qui entraîne la production de métabolites secondaires uniques. L'un des métabolites secondaires les plus fréquemment produits est l'acide usnique, dont on croit qu'il est produit par deux gènes. Les objectifs de la présente étude consistaient à comparer l'expression génique de la polycétide synthase (PKS) et la concentration d'acide usnique chez Cladonia uncialis (L.) Weber ex F.H. Wigg., en fonction de deux facteurs environnementaux. Soixante-quinze échantillons de lichen ont été recueillis à trois endroits à Terre-Neuve à l'aide d'une méthode de transects en bandes (X 5transectsX 5 quadrats). La concentration d'acide usnique a été mesurée par chromatographie liquide avec spectrométrie de masse en tandem et l'expression génique de deux gènes de PKS (la méthylphloracétophénone oxydase MPAO et la méthylphloracétophénone synthase MPAS) a été examinée par PCR quantitative en temps réel. Les résultats ont montré que le pourcentage de couverture végétale de C. uncialis était affecté par le pH du sol, mais pas par l'humidité du sol alors que la concentration d'acide usnique n'était pas affectée par le pH ou l'humidité du sol. Le niveau d'expression du gène MMPAO était significativement affecté par le pH du sol, mais pas par l'humidité du sol alors que le niveau d'expression du gène MPAS n'était pas affecté par le pH du sol ou par l'humidité du sol. Il n'y avait pas de relation significative entre les niveaux d'expression des gènes MPAS et MPAO et la concentration d'acide usnique. Ces résultats suggèrent que le pH du sol peut être important pour la production d'acide usnique par C. uncialis, mais les gènes impliqués nécessitent une étude plus approfondie. [Traduit par la Rédaction] Mots-clés : diversité des lichens, MPAO, MPAS, pourcentage de couverture, métabolites secondaires, teneur en humidité du sol, pH du sol., Introduction Lichens are intimate symbioses, which result from the association between fungi (mycobionts) and green algae or cyanobacteria (photobionts). Because lichens absorb water and nutrients through the surface of the [...]

An understanding of the biochemistry of the giant cell tumour of bone (GCTB) provides an opportunity for the development of prognostic markers and identification of therapeutic targets. Based on metabolomic analysis, we proposed glycerophospholipid metabolism as the altered pathway in GCTB., The objective of this study was to identify these altered metabolites. Using phosphorus-31 nuclear magnetic resonance spectroscopy ([.sup.31]P-NMR), sphingomyelin was determined to be the most dysregulated phospholipid in tissue samples from six patients with GCTB. Enzymes related to its biosynthesis and hydrolysis were examined using immunodetection techniques. High expression of sphingomyelin synthases 1 and 2, but low expression of neutral sphingomyelinase 2 (nSMase2) was found in GCTB tissues compared to non-neoplastic bone tissues. Sphingomyelin/ceramide biosynthesis is dysregulated in GCTB due to alterations in the expression of SMS1, SMS2, and nSMase2. Key words: giant cell tumour of bone, sphingomyelin, ceramide, sphingomyelin synthase, sphingomyelinase, neutral sphingomyelinase 2, bone cancer, giant cell-rich bone tumours, 31P-NMR, glycerophospholipids. La compréhension de la biochimie de la tumeur à cellules géantes de l'os (TCG) offre la possibilité de développer des marqueurs pronostiques et d'identifier des cibles thérapeutiques. En se fondant sur une analyse métabolomique, les auteurs ont proposé que le métabolisme des glycérophospholipides soit la voie affectée dans la TCG. L'objectif de cette étude était d'identifier ces métabolites touchés. Grâce à la spectroscopie par résonance magnétique nucléaire au phosphore 31 ([.sup.31]P-NMR), la sphingomyéline a été identifiée comme étant le phospholipide dont la dérégulation était la plus importante dans les échantillons de tissus de six patients atteints de TCG ; les enzymes liées à sa biosynthèse et à son hydrolyse ont ensuite été examinées à l'aide de techniques d'immunodétection. Une expression élevée des sphingomyéline synthases 1 et 2, mais une faible expression de la sphingomyélinase neutre 2 (nSMase2), ont été trouvées dans les tissus de TCG comparativement aux tissus osseux non néoplasiques. La biosynthèse de sphingomyéline et de céramide est dérégulée dans la TCG en raison de modifications de l'expression de SMS1, SMS2 et nSMase2. Mots-clés : tumeur à cellules géantes de l'os, sphingomyéline, céramide, sphingomyéline synthase, sphingomyélinase, sphingomyélinase neutre 2, cancer de l'os, tumeurs osseuses riches en cellules géantes, 31P-RMN, glycérophospholipides., Introduction Giant cell tumour of bone (GCTB) is one of the most prevalent epiphyseal and meta-epiphyseal neoplasms, which are locally aggressive, recurrent, and have high metastatic potential to the lung [...]

Cholesterol is an essential lipid and its synthesis is nutritionally and energetically costly.sup.1,2. In mammals, cholesterol biosynthesis increases after feeding and is inhibited under fasting conditions.sup.3. However, the regulatory mechanisms of cholesterol biosynthesis at the fasting-feeding transition remain poorly understood. Here we show that the deubiquitylase ubiquitin-specific peptidase 20 (USP20) stabilizes HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthetic pathway, in the feeding state. The post-prandial increase in insulin and glucose concentration stimulates mTORC1 to phosphorylate USP20 at S132 and S134; USP20 is recruited to the HMGCR complex and antagonizes its degradation. The feeding-induced stabilization of HMGCR is abolished in mice with liver-specific Usp20 deletion and in USP20(S132A/S134A) knock-in mice. Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet-induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure. These metabolic changes are reversed by expression of the constitutively stable HMGCR(K248R). This study reveals an unexpected regulatory axis from mTORC1 to HMGCR via USP20 phosphorylation and suggests that inhibitors of USP20 could be used to lower cholesterol levels to treat metabolic diseases including hyperlipidaemia, liver steatosis, obesity and diabetes. mTORC1 stabilizes HMG-CoA reductase, a rate-limiting enzyme in the cholesterol biosynthesis pathway, via the deubiquitylase USP20 in response to feeding., Author(s): Xiao-Yi Lu [sup.1] , Xiong-Jie Shi [sup.1] , Ao Hu [sup.1] , Ju-Qiong Wang [sup.1] , Yi Ding [sup.1] , Wei Jiang [sup.1] , Ming Sun [sup.1] , Xiaolu [...]

The microphthalmia family of transcription factors (MiT/TFEs) controls lysosomal biogenesis and is negatively regulated by the nutrient sensor mTORC1. However, the mechanisms by which cells with constitutive mTORC1 signaling maintain lysosomal catabolism remain to be elucidated. Using the murine epidermis as a model system, we found that epidermal Tsc1 deletion resulted in a phenotype characterized by wavy hair and curly whiskers, and was associated with increased EGFR and HER2 degradation. Unexpectedly, constitutive mTORC1 activation with Tsc1 loss increased lysosomal content via upregulated expression and activity of MiT/TFEs, whereas genetic deletion of Rheb or Rptor or prolonged pharmacologic mTORC1 inactivation had the reverse effect. This paradoxical increase in lysosomal biogenesis by mTORC1 was mediated by feedback inhibition of AKT, and a resulting suppression of AKT-induced MiT/TFE downregulation. Thus, inhibiting hyperactive AKT signaling in the context of mTORC1 loss-of-function fully restored MiT/TFE expression and activity. These data suggest that signaling feedback loops work to restrain or maintain cellular lysosomal content during chronically inhibited or constitutively active mTORC1 signaling, respectively, and reveal a mechanism by which mTORC1 regulates upstream receptor tyrosine kinase signaling., Introduction The microphthalmia family of transcription factors (MiT/TFEs) is composed of 4 conserved members (Mitf/Tfe3/Tfeb/Tfec) that are essential regulators of lysosomal biogenesis and autophagy. MiTs are functionally redundant and regulate [...]

Pericyclic reactions are powerful transformations for the construction of carbon-carbon and carbon-heteroatom bonds in organic synthesis. Their role in biosynthesis is increasingly apparent, and mechanisms by which pericyclases can catalyse reactions are of major interest.sup.1. [4+2] cycloadditions (Diels-Alder reactions) have been widely used in organic synthesis.sup.2 for the formation of six-membered rings and are now well-established in biosynthesis.sup.3-6. [6+4] and other 'higher-order' cycloadditions were predicted.sup.7 in 1965, and are now increasingly common in the laboratory despite challenges arising from the generation of a highly strained ten-membered ring system.sup.8,9. However, although enzyme-catalysed [6+4] cycloadditions have been proposed.sup.10-12, they have not been proven to occur. Here we demonstrate a group of enzymes that catalyse a pericyclic [6+4] cycloaddition, which is a crucial step in the biosynthesis of streptoseomycin-type natural products. This type of pericyclase catalyses [6+4] and [4+2] cycloadditions through a single ambimodal transition state, which is consistent with previous proposals.sup.11,12. The [6+4] product is transformed to a less stable [4+2] adduct via a facile Cope rearrangement, and the [4+2] adduct is converted into the natural product enzymatically. Crystal structures of three pericyclases, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis establish the mechanism of this transformation. This work shows how enzymes are able to catalyse concerted pericyclic reactions involving ambimodal transition states. Enzymatic catalysis of pericyclic [6+4] cycloaddition reactions to form ten-membered rings is observed during biosynthesis of the macrocyclic antibiotic streptoseomycin, and the mechanism of these transformations is established., Author(s): Bo Zhang [sup.1] , Kai Biao Wang [sup.1] , Wen Wang [sup.1] , Xin Wang [sup.2] , Fang Liu [sup.2] , Jiapeng Zhu [sup.3] , Jing Shi [sup.1] , [...]

Camptotheca acuminata Decne (Nyssaceae) is major natural source of camptothecin, an anticancer drug widely used for clinic therapy. Previous works have shown that many plant hormones or elicitors could regulate camptothecin biosynthesis, but few reports have examined sustainable effects of these plant hormones on plants for producing camptothecin. In this work, seedlings obtained from in vitro rapid propagation were used for investigating the sustainable effect of salicylic acid (SA) on transplanted C. acuminata tissues, especially leaves, for camptothecin biosynthesis. Our results indicate that exogenous SA could continuously induce the expression of iridoids pathway genes in C. acuminata leaves for promoting camptothecin production. High expression of iridoids pathway genes still could be observed in bioactive young leaves 180 days after seedlings had been transplanted to soil. In C. acuminata, iridoids pathway genes are expressed at very low levels or are not detectable in old leaves, resulting in decreasing production of camptothecin with leaf development. Interestingly, the expression of these genes could be detected clearly in old leaves of transplanted C. acuminata pretreated with 10 [micro]mol x [L.sup.-1] of SA during the in vitro rapid propagation phase, which indicates the particular function of SA for a sustainable effect on maintaining relatively high expression levels of iridoids pathway genes for camptothecin biosynthesis.Key words: Camptotheca acuminata Decne, camptothecin, salicylic acid, in vitro rapid propagation, iridoids biosynthesis.Camptotheca acuminata Decne (Nyssacees) est une source naturelle importante de camptothecine, un medicament contre le cancer largement utilise en therapie clinique. Des travaux anterieurs ont montre que plusieurs hormones ou eliciteurs vegetaux peuvent reguler la biosynthese de la camptothecine mais peu d'entre eux ont porte sur l'effet durable de ces hormones sur les plantes pour la production de camptothecine. L'acide salicylique (AS) est une hormone vegetale fonctionnelle qui pourrait reguler plusieurs processus physiologiques et biochimiques chez les plantes et promouvoir la production de camptothecine chez C. acuminata. Dans ce travail, des semis obtenus par propagation rapide in vitro ont ete utilises pour etudier l'effet durable de l'AS sur des tissus transplantes de C. acuminata, particulierement les feuilles, pour la biosynthese de camptothecine. Nos resultats indiquent que l'AS exogene peut induire de facon continue l'expression des genes du sentier des iridoides dans les feuilles de C. acuminata et promouvoir la production de camptothecine. Apres avoir ete transplantees dans le sol depuis 180 jours, une forte expression des genes du sentier des iridoides pouvait encore etre observee dans les jeunes feuilles bioactives. Chez C. acuminata, les genes du sentier des iridoides sont exprimes a niveaux tres faibles ou indetectables chez les vieilles feuilles, ce qui entraine une production insuffisante de camptothecine a mesure que les feuilles se developpent. Il est interessant de noter que l'expression de ces genes pouvait clairement etre detectee dans les vieilles feuilles transplantees de C. acuminata qui avaient ete pretraitees avec 10 [micro]mol x [L.sup.-1] d'AS durant la phase de propagation rapide in vitro, ce qui montre que l'AS a une fonction particuliere a cause de son effet durable sur le maintien de l'expression, a niveaux relativement eleves, des genes du sentier des iridoides qui mene a la biosynthese de la camptothecine. [Traduit par la Redaction]Mots-cles : Camptotheca acuminata Decne, camptothecine, acide salicylique, propagation rapide in vitro, biosyntheses des iridoides., 1. IntroductionCamptothecin is a water-insoluble terpenoid indole alkaloid (TIA) that was originally isolated from the extract of Camptotheca acuminate Decne (Nyssacees), a traditional Chinese medicinal tree. Further investigation showed that [...]

Background: Changes in cholesterol metabolism are common hallmarks of neurodevelopmental pathologies. A diverse array of genetic disorders of cholesterol metabolism support this claim as do multiple lines of research that demonstrate chemical inhibition of cholesterol biosynthesis compromises neurodevelopment. Recent work has revealed that a number of commonly used pharmaceuticals induce changes in cholesterol metabolism that are similar to changes induced by genetic disorders with devastating neurodevelopmental deficiencies. OBJECTIVES: We tested the hypothesis that common environmental toxicants may also impair cholesterol metabolism and thereby possibly contribute to neurodevelopmental toxicity. Methods: Using high-throughput screening with a targeted lipidomic analysis and the mouse neuroblastoma cell line, Neuro-2a, the ToxCast[TM] chemical library was screened for compounds that impact sterol metabolism. Validation of chemical effects was conducted by assessing cholesterol biosynthesis in human induced pluripotent stem cell (hiPSC)-derived neuroprogenitors using an isotopically labeled cholesterol precursor and by monitoring product formation with UPLC-MS/MS. Results: Twenty-nine compounds were identified as validated lead-hits, and four were prioritized for further study (endosulfan sulfate, tributyltin chloride, fenpropimorph, and spiroxamine). All four compounds were validated to cause hypocholesterolemia in Neuro-2a cells. The morpholine-like fungicides, fenpropimorph and spiroxamine, mirrored their Neuro-2a activity in four immortalized human cell lines and in a human neuroprogenitor model derived from hiPSCs, but endosulfan sulfate and tributyltin chloride did not. Conclusions: These data reveal the existence of environmental compounds that interrupt cholesterol biosynthesis and that methodologically hiPSC neuroprogenitor cells provide a particularly sensitive system to monitor the effect of small molecules on de novo cholesterol formation. https://doi.org/10.1289/EHP5053, Introduction Human exposure studies with organic solvents, metal exposures, and air pollutants have revealed the existence of developmental neurotoxicants (Costa et al. 2014; Grandjean and Landrigan 2006, 2014). To proactively [...]

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA-BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate--and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a 'finger helix' that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves 'up' and 'down' in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA's transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation., Cellulose is an abundant structural cell component produced by many organisms, including bacteria, vascular plants and animals (1-4). It is a linear polymer of glucose molecules joined between their C1 [...]

Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) is the major cause of cystic fibrosis, one of the most common inherited childhood diseases. The mutated CFTR anion channel is not fully glycosylated and shows minimal activity in bronchial epithelial cells of patients with cystic fibrosis. Low temperature or inhibition of histone deacetylases can partly rescue ΔF508 CFTR cellular processing defects and function. A favourable change of ΔF508 CFTR protein-protein interactions was proposed as a mechanism of rescue; however, CFTR interactome dynamics during temperature shift and inhibition of histone deacetylases are unknown. Here we report the first comprehensive analysis of the CFTR and ΔF508 CFTR interactome and its dynamics during temperature shift and inhibition of histone deacetylases. By using a novel deep proteomic analysis method, we identify 638 individual highconfidence CFTR interactors and discover a ΔF508 deletion-specific interactome, which is extensively remodelled upon rescue. Detailed analysis of the interactome remodelling identifies key novel interactors, whose loss promote ΔF508 CFTR channel function in primary cystic fibrosis epithelia or which are critical for CFTR biogenesis. Our results demonstrate that global remodelling of ΔF508 CFTR interactions is crucial for rescue, and provide comprehensive insight into the molecular disease mechanisms of cystic fibrosis caused by deletion of F508., Cystic fibrosis (CF) is one of the most common inherited childhood diseases, with about 10 million carriers in the USA alone. The disease is caused by mutation of the CFTR [...]