Ketogenic diets attenuate cyclooxygenase and lipoxygenase gene expression in multiple sclerosis

Background: Adapted ketogenic diet (AKD) and caloric restriction (CR) have been suggested as alternative therapeutic strategies for inflammatory, hyperproliferative and neurodegenerative diseases. Pro-inflammatory eicosanoids have been implicated in the pathogenesis of multiple sclerosis since they augment vascular permeability and induce leukocyte migration into the brain. We explored the impact of ketogenic diets on gene expression of biosynthetic enzymes for pro- (ALOX5, COX1, COX2) and anti-inflammatory (ALOX15) eicosanoids in patients with relapsing-remitting multiple sclerosis. Methods: 60 adults were prospectively recruited for this six months randomized controlled trial and the impact of dietary treatment on the Multiple Sclerosis Quality of Life-54 index (ClinicalTrials.gov (NCT01538355) has previously been published. Here we explored 24 patients (8 controls, 5 on CR and 11 on AKD). For statistical analysis we combined the two diet groups to a single pooled treatment group. Findings: Inter-group comparison indicated that expression of the pro-inflammatory ALOX5 in the pooled treatment group was significantly (p <0.05) reduced when compared with the control group. Moreover, intra-group comparison (same individuals before and after dietary treatment) suggested significantly impaired expression of other pro-inflammatory enzymes, such as COX1 (p < 0.001) and COX2 ( p < 0.05). Finally, pretreatment cross-group analysis revealed a significant positive correlation between expression of pro-inflammatory ALOX5 and COX2 and an inverse correlation of ALOX5 and COX1 expression with the MSQoL-54 index. Interpretation: Ketogenic diets can reduce the expression of enzymes involved in the biosynthesis of pro-inflammatory eicosanoids. Pharmacological interference with eicosanoid biosynthesis might constitute a strategy supplementing current therapeutic approaches for MS. (C) 2018 The Authors. Published by Elsevier B.V

Male guanine-rich RNA sequence binding factor 1 knockout mice (Grsf1−/−) gain less body weight during adolescence and adulthood

The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the heterogenous nuclear ribonucleoprotein H/F (hnRNP H/F) family that binds to guanine-rich RNA sequences forming G-quadruplex structures. In mice and humans there are single copy GRSF1 genes, but multiple transcripts have been reported. GRSF1 has been implicated in a number of physiological processes (e.g. embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of viral infections and hyperproliferative diseases. These postulated biological functions of GRSF1 originate from in vitro studies rather than complex in vivo systems. To assess the in vivo relevance of these findings, we created systemic Grsf1(-/-) knockout mice lacking exons 4 and 5 of the Grsf1 gene and compared the basic functional characteristics of these animals with those of wildtype controls. We found that Grsf1-deficient mice are viable, reproduce normally and have fully functional hematopoietic systems. Up to an age of 15 weeks they develop normally but when male individuals grow older, they gain significantly less body weight than wildtype controls in a gender-specific manner. Profiling Grsf1 mRNA expression in different mouse tissues we observed high concentrations in testis. Comparison of the testicular transcriptomes of Grsf1(-/-) mice and wildtype controls confirmed near complete knock-out of Grsf1 but otherwise subtle differences in transcript regulations. Comparative testicular proteome analyses suggested perturbed mitochondrial respiration in Grsf1(-/-) mice which may be related to compromised expression of complex I proteins. Here we present, for the first time, an in vivo complete Grsf1 knock-out mouse with comprehensive physiological, transcriptomic and proteomic characterization to improve our understanding of the GRSF1 beyond in vitro cell culture models

Infection of cells with replication deficient adenovirus induces cell cycle alterations and leads to downregulation of E2F-1

AbstractGene products of recombinant replication-deficient adenovirus vectors of the first generation (Ad vector) can induce cell cycle dysregulation and apoptosis after infection in eukaryotic cells. The mechanisms underlying this complex process are largely unknown. Therefore, we investigated the regulation of the pRb/E2F-1 complex, which controls transition from G0/G1 to S phase of the cell cycle. As Ad vector infection results in a decrease in the number of cells in G0/G1 phase of the cell cycle, we observed a decline of the pRb protein level and, surprisingly, also a decrease of the E2F-1 protein and mRNA level in infected cell lines. Furthermore, in contrast to the reduction of cells in the G0/G1 phase we observed increased protein levels of p53 and p21 proteins. However, as experiments in p53 deficient cell lines indicated, the decrease of pRb and E2F-1 is independent of p53 and p21 expression. Moreover, results obtained with Rb deficient cell lines indicated that the reduced E2F-1 expression is independent of pRb. These results suggest that Ad vector-induced cell cycle dysregulation is associated with a specific downregulation of E2F-1 independent of Rb and p53 genomic status of cells

The Reaction Specificity of Mammalian ALOX15 Orthologs is Changed During Late Primate Evolution and These Alterations Might Offer Evolutionary Advantages for Hominidae

Arachidonic acid lipoxygenases (ALOXs) have been implicated in the immune response of mammals. The reaction specificity of these enzymes is decisive for their biological functions and ALOX classification is based on this enzyme property. Comparing the amino acid sequences and the functional properties of selected mammalian ALOX15 orthologs we previously hypothesized that the reaction specificity of these enzymes can be predicted based on their amino acid sequences (Triad Concept) and that mammals, which are ranked in evolution below gibbons, express arachidonic acid 12-lipoxygenating ALOX15 orthologs. In contrast, Hominidae involving the great apes and humans possess 15-lipoxygenating enzymes (Evolutionary Hypothesis). These two hypotheses were based on sequence data of some 60 mammalian ALOX15 orthologs and about half of them were functionally characterized. Here, we compared the ALOX15 sequences of 152 mammals representing all major mammalian subclades expressed 44 novel ALOX15 orthologs and performed extensive mutagenesis studies of their triad determinants. We found that ALOX15 genes are absent in extant Prototheria but that corresponding enzymes frequently occur in Metatheria and Eutheria. More than 90% of them catalyze arachidonic acid 12-lipoxygenation and the Triad Concept is applicable to all of them. Mammals ranked in evolution above gibbons express arachidonic acid 15-lipoxygenating ALOX15 orthologs but enzymes with similar specificity are only present in less than 5% of mammals ranked below gibbons. This data suggests that ALOX15 orthologs have been introduced during Prototheria-Metatheria transition and put the Triad Concept and the Evolutionary Hypothesis on a much broader and more reliable experimental basis

Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells

Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S–hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type

Unbalanced expression of glutathione peroxidase 4 and arachidonate 15-lipoxygenase affects acrosome reaction and In Vitro fertilization

Glutathione peroxidase 4 (Gpx4) and arachidonic acid 15 lipoxygenase (Alox15) are counterplayers in oxidative lipid metabolism and both enzymes have been implicated in spermatogenesis. However, the roles of the two proteins in acrosomal exocytosis have not been explored in detail. Here we characterized Gpx4 distribution in mouse sperm and detected the enzyme not only in the midpiece of the resting sperm but also at the anterior region of the head, where the acrosome is localized. During sperm capacitation, Gpx4 translocated to the post-acrosomal compartment. Sperm from Gpx4+/Sec46Ala mice heterozygously expressing a catalytically silent enzyme displayed an increased expression of phosphotyrosyl proteins, impaired acrosomal exocytosis after in vitro capacitation and were not suitable for in vitro fertilization. Alox15-deficient sperm showed normal acrosome reactions but when crossed into a Gpx4-deficient background spontaneous acrosomal exocytosis was observed during capacitation and these cells were even less suitable for in vitro fertilization. Taken together, our data indicate that heterozygous expression of a catalytically silent Gpx4 variant impairs acrosomal exocytosis and in vitro fertilization. Alox15 deficiency hardly impacted the acrosome reaction but when crossed into the Gpx4-deficient background spontaneous acrosomal exocytosis was induced. The detailed molecular mechanisms for the observed effects may be related to the compromised redox homeostasis

Novel Genetic Risk and Metabolic Signatures of Insulin Signaling and Androgenesis in the Anovulation of Polycystic Ovary Syndrome

Funding Information: The authors are grateful to all staff in the PCOSAct group for their effort in the collection of blood samples and clinical dataset which used in current study. Special thanks to Prof. Attila Toth from Institute of Physiological Chemistry, Dresden, Germany for the REC114 antibody. This study was supported by the National key Research and Development Program of China (2019YFC1709500); the National Collaboration Project of Critical Illness by Integrating Chinese Medicine and Western Medicine; the Project of Heilongjiang Province Innovation Team “TouYan;” the Yi-Xun Liu and Xiao-Ke Wu Academician Workstation; the Innovation Team of Reproductive Technique with Integrative Chinese Medicine and Western Medicine in Xuzhou City, China; Heilongjiang University of Chinese Medicine from the National Clinical Trial Base; Heilongjiang Provincial Clinical Research Center for Ovary Diseases; the Research Grant Council (T13-602/21-N, C5045-20EF, and 14122021); and Food and Health Bureau in Hong Kong, China (06171026). Ben Willem J. Mol is supported by a National Health and Medical Research Council (NHMRC) Investigator grant (GNT1176437). Ben Willem J. Mol reports consultancy for ObsEva and Merck and travel support from Merck. Xiaoke Wu, Yongyong Shi, and Chi Chiu Wang developed the research question and designed the study. Xiaoke Wu, Yongyong Shi, Yijuan Cao, and Chi Chiu Wang designed the analysis. Yongyong Shi and Zhiqiang Li contributed to the design of the experiment of whole-exome plus targeted SNP sequencing and the analysis, and interpreted the results. Jingshu Gao, Hui Chang, Duojia Zhang, Jing Cong, Yu Wang, Qi Wu, Xiaoxiao Han, Pui Wah Jacqueline Chung, Yiran Li, and Lin Zeng contributed to the experiment of metabolic profile and immunofluorescent staining and the analysis, and interpreted the results. Astrid Borchert and Hartmut Kuhn provided antibody support and advice. Xu Zheng and Lingxi Chen contributed to create the predictive model with deep machine learning. Jian Li, Qi Wu, Hongli Ma, Xu Zheng, and Lingxi Chen contributed to the analysis of the clinical characteristics and interpreted the results. Jian Li, Hongli Ma, Hui Chang, Jing Cong, and Chi Chiu Wang drafted the manuscript. All authors reviewed and revised the manuscript. Xiaoke Wu is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Xiaoke Wu, Chi Chiu Wang, Yijuan Cao, Jian Li, Zhiqiang Li, Hongli Ma, Jingshu Gao, Hui Chang, Duojia Zhang, Jing Cong, Yu Wang, Qi Wu, Xiaoxiao Han, Pui Wah Jacqueline Chung, Yiran Li, Xu Zheng, Lingxi Chen, Lin Zeng, Astrid Borchert, Hartmut Kuhn, Zijiang Chen, Ernest Hung Yu Ng, Elisabet Stener-Victorin, Heping Zhang, Richard S. Legro, Ben Willem J. Mol, and Yongyong Shi declare that they have no conflict of interest or financial conflicts to disclose. Funding Information: This study was supported by the National key Research and Development Program of China ( 2019YFC1709500 ); the National Collaboration Project of Critical Illness by Integrating Chinese Medicine and Western Medicine ; the Project of Heilongjiang Province Innovation Team “TouYan;” the Yi-Xun Liu and Xiao-Ke Wu Academician Workstation; the Innovation Team of Reproductive Technique with Integrative Chinese Medicine and Western Medicine in Xuzhou City , China; Heilongjiang University of Chinese Medicine from the National Clinical Trial Base ; Heilongjiang Provincial Clinical Research Center for Ovary Diseases ; the Research Grant Council ( T13-602/21-N , C5045-20EF , and 14122021 ); and Food and Health Bureau in Hong Kong, China ( 06171026 ). Publisher Copyright: © 2023Peer reviewedPublisher PD