CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress

Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Biosynthesis of Long-chain Fatty Acid Amides

The vast variety of long-chain fatty acid amides identified in biological systems is intriguing. The general structure of a fatty acid amide is R-CO-NH-X, where R is an alkyl group and X is derived from an immense variety of biogenic amines. Although structurally simple, the bioactivities of these molecules as signaling lipids are very diverse and have just recently begun to emerge in the literature. Interest in the long-chain fatty acid amides dramatically increased following the identification and characterization of one specific N-acylethanolamine, N-arachidonoylethanolamine (anandamide), as the endogenous ligand for the cannabinoid receptors in the mammalian brain. Since this discovery, the details of N-acylethanolamine metabolism have been elucidated. However, a lesser extent of progress has been made in the last twenty years to identify and study the non-N-acylethanolamine long-chain fatty acid amides. The focus of this dissertation is the elucidation of the biosynthetic pathways for long-chain fatty acid amides, including N-acylglycines, primary fatty acid amides, N-acylarylalkylamides, and N-acylethanolamines. The details of long-chain fatty acid amide metabolism will lead to the determination of possible therapeutic targets. We identified mammalian glycine N-acyltransferase like 3 as the enzyme that catalyzes the formation of long-chain N-acylglycines in mouse N18TG2 neuoblastoma cells, identified and quantified a panel of long-chain fatty acid amides in Drosophila melanogaster extracts by LC/QTOF-MS, established Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism, and identified arylalkylamine N-acyltransferase like 2 as the enzyme that catalyzes the formation of long-chain N-acylserotonins and N-acyldopamines in Drosophila melanogaster

Soil Amendment and Storage Effect the Quality of Winter Melons (<i>Benincasa hispida</i> (Thunb) Cogn.) and Their Juice

Winter melon fruits were grown in the field using anaerobic soil disinfestation (ASD) and conventional fertilizer alone as the control treatment. Fruits were harvested and stored at 20 °C for 120 d, the juice was processed on day one and day 120, and the effects of soil amendment and 120 d storage on the juice’s physical and chemical (sugars, acids, volatile and nutritional compounds) properties were evaluated. Fruit juice extracted from ASD-grown fruit had greater magnitude of zeta potential than the control juice, indicating it was physically more stable than the juice obtained from the control conditions. ASD fruit juice had lower soluble solids content (SSC), and lower volatile compounds that contribute green, grass, and sulfur notes, and negatively influence flavor quality. ASD fruit juice had higher vitamin B5 and cytidine. Juice processed from 120 d stored fruit had less yield due to 12.4–15.6% weight loss. The non-soluble solids content was higher and particle size was larger, and the SSC and individual sugars decreased. However, titratable acidity (TA) increased primarily due to increased citric acid. Out of 16 free amino acids, 6 increased and only 1 decreased. However, three out of five nucleosides decreased; vitamins B1 and B6 increased; vitamins B2, B3 and C decreased. Overall, juice derived from fruit produced using ASD was physically more stable and had less SSC and off-odor volatiles than the control, while the fruit juice of those stored for 120 d had lower SSC and higher TA and nutritional profiles, comparable to freshly harvested fruit

Mechanistic and Structural Analysis of a \u3cem\u3eDrosophila melanogaster\u3c/em\u3e Enzyme, Arylalkylamine \u3cem\u3eN\u3c/em\u3e-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of \u3cem\u3eN\u3c/em\u3e-Acetylarylalkylamides and \u3cem\u3eN\u3c/em\u3e-Acetylhistamine

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of \u3e10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH–activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis

Effect of Simulation Role on Clinical Decision-Making Accuracy

Background: Clinical simulation affords valuable practice with the process of clinical decision-making. Participation in the simulation experience often includes various roles (e.g., primary nurse; observer) as well as familiar and unfamiliar situations. Method: This study used a quantitative, mixed factorial design. Results: Role did not impact clinical decision-making accuracy on the familiar situation. On the unfamiliar situation, observers outperformed other roles, whereas family members were markedly less accurate. Conclusion: These results emphasize the need for students to rotate through various roles and for educators to consider the nature and goal of the scenario when deciding what types of roles to assign

Glycine N-acyltransferase-like 3 is Responsible for Long-chain N-acylglycine Formation in N\u3csub\u3e18\u3c/sub\u3eTG\u3csub\u3e2\u3c/sub\u3e Cells

Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [(13)C18]N-oleoylglycine and decreased levels of [(13)C18]oleamide when the N18TG2 cells were grown in the presence of [(13)C18]oleic acid. The addition of [1-(13)C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-(13)C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein

Mechanistic and Structural Analysis of a <i>Drosophila melanogaster</i> Enzyme, Arylalkylamine <i>N</i>‑Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of <i>N</i>‑Acetylarylalkylamides and <i>N</i>‑Acetylhistamine

Arylalkylamine <i>N</i>-acetyltransferase like 7 (AANATL7) catalyzes the formation of <i>N</i>-acetylarylalkylamides and <i>N</i>-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related <i>N</i>-acetyltransferases, many members being linked to important roles in both human metabolism and disease. <i>Drosophila melanogaster</i> utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified <i>D. melanogaster</i> AANATL7 in <i>Escherichia coli</i> and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because <i>D. melanogaster</i> has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH–activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a p<i>K</i>_a of 7.1 that is assigned to Glu-26 as a general base and a second p<i>K</i>_a of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis