Retinal metabolic events in preconditioning light stress as revealed by wide-spectrum targeted metabolomics

Introduction

Light is the primary stimulus for vision, but may also cause damage to the retina. Pre-exposing the retina to sub-lethal amount of light (or preconditioning) improves chances for retinal cells to survive acute damaging light stress.

Objectives

This study aims at exploring the changes in retinal metabolome after mild light stress and identifying mechanisms that may be involved in preconditioning.

Methods

Retinas from 12 rats exposed to mild light stress (1000 lux?×?for 12 h) and 12 controls were collected one and seven days after light stress (LS). One retina was used for targeted metabolomics analysis using the Biocrates p180 kit while the fellow retina was used for histological and immunohistochemistry analysis.

Results

Immunohistochemistry confirmed that in this experiment, a mild LS with retinal immune response and minimal photoreceptor loss occurred. Compared to controls, LS induced an increased concentration in phosphatidylcholines. The concentration in some amino acids and biogenic amines, particularly those related to the nitric oxide pathway (like asymmetric dimethylarginine (ADMA), arginine and citrulline) also increased 1 day after LS. 7 days after LS, the concentration in two sphingomyelins and phenylethylamine was found to be higher. We further found that in controls, retina metabolome was different between males and females: male retinas had an increased concentration in tyrosine, acetyl-ornithine, phosphatidylcholines and (acyl)-carnitines.

Conclusions

Besides retinal sexual metabolic dimorphism, this study shows that preconditioning is mostly associated with re-organisation of lipid metabolism and changes in amino acid composition, likely reflecting the involvement of arginine-dependent NO signalling.

     

Effects of resveratrol on HepG2 cells as revealed by H-NMR based metabolic profiling

Background

Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis.

Methods

We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a ¹H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways.

Results

Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation.

Conclusions

Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation.

General significance

NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.     

Distinct genomic traits of acral and mucosal melanomas revealed by targeted mutational profiling

The incidence of melanoma is rising globally including China. Comparing to Caucasians, the incidence of non‐cutaneous melanomas is significantly higher in Chinese. Herein, we performed genomic profiling of 89 Chinese surgically resected primary melanomas, including acral (n = 54), cutaneous (n = 22), and mucosal (n = 13), by hybrid capture‐based next‐generation sequencing. We show that mucosal melanomas tended to harbor more pathogenic mutations than other types of melanoma, though the biological significance of this finding remains uncertain. Chromosomal arm‐level alterations including 6q, 9p, and 10p/q loss were highly recurrent in all subtypes, but mucosal melanoma was significantly associated with increased genomic instability. Importantly, 7p gain significantly correlated with unfavorable clinical outcomes in non‐cutaneous melanomas, representing an intriguing prognostic biomarker of those subtypes. Furthermore, focal amplification of 4q12 (KIT, KDR, and PDGFRα) and RAD51 deletion were more abundant in mucosal melanoma, while NOTCH2 amplification was enriched in acral melanoma. Additionally, cutaneous melanomas had higher mutation load than acral melanomas, while mucosal melanomas did not differ from other subtypes in mutation burden. Together, our data revealed important features of acral and mucosal melanomas in Chinese including distinctive driver mutation pattern and increased genomic instability. These findings highlight the possibilities of combination therapies in the clinical management of melanoma.     

Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling.

Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3'-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.     

A second target of the antimalarial and antibacterial agent fosmidomycin revealed by cellular metabolic profiling

Antimicrobial drug resistance is an urgent problem in the control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies for the treatment of malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and Escherichia coli bacteria: in both organisms, metabolic profiling demonstrated a block of isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent for blocking non-mevalonate isoprenoid metabolism and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.     

Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.     

Attenuated metabolism is a hallmark of obesity as revealed by comparative proteomic analysis of human omental adipose tissue

Obesity is recognized as an epidemic health problem worldwide. In humans, the accumulation of omental rather than subcutaneous fat appears to be tightly linked to insulin resistance, type 2 diabetes and cardiovascular disease. Differences in gene expression profiles in the adipose tissue comparing non-obese and obese subjects have been well documented. However, to date, no comparative proteomic studies based on omental fat have investigated the influence of obesity in protein expression. In this work, we searched for proteins differentially expressed in the omental fat of non-obese and obese subjects using 2D-DIGE and MS. Forty-four proteins, several of which were further studied by immunoblotting and immunostaining analyses, showed significant differences in the expression levels in the two groups of subjects. Our findings reveal a clearly distinctive proteomic profile between obese and non-obese subjects which emphasizes: i) reduced metabolic activity in the obese fat, since most down-regulated proteins were engaged in metabolic pathways; and ii) morphological and structural cell changes in the obese fat, as revealed by the functions exerted by most up-regulated proteins. Interestingly, transketolase and aminoacylase-1 represent newly described molecules involved in the pathophysiology of obesity, thus opening up new possibilities in the study of obesity.     

Methionine adenosyltransferase as a useful molecular systematics tool revealed by phylogenetic and structural analyses

Structural and phylogenetic relationships among Bacteria and Eukaryota were analyzed by examining 292 methionine adenosyltransferase (MAT) amino acid sequences with respect to the crystal structure of this enzyme established for Escherichia coli and rat liver. Approximately 30% of MAT residues were found to be identical in all species. Five highly conserved amino acid sequence blocks did not vary in the MAT family. We detected specific structural features that correlated with sequence signatures for several clades, allowing taxonomical identification by sequence analysis. In addition, the number of amino acid residues in the loop connecting beta-strands A2 and A3 served to clearly distinguish sequences between eukaryotes and eubacteria. The molecular phylogeny of MAT genes in eukaryotes can be explained in terms of functional diversification coupled to gene duplication or alternative splicing and adaptation through strong structural constraints. Sequence analyses and intron/exon junction positions among nematodes, arthropods and vertebrates support the traditional Coelomata hypothesis. In vertebrates, the liver MAT I isoenzyme has gradually adapted its sequence towards one providing a more specific liver function. MAT phylogeny also served to cluster the major bacterial groups, demonstrating the superior phylogenetic performance of this ubiquitous, housekeeping gene in reconstructing the evolutionary history of distant relatives.     

Evolutionary history of the calcitonin gene-related peptide family in vertebrates revealed by comparative genomic analyses

The calcitonin gene-related peptide (CGRP) family is composed of CGRP, amylin and adrenomedullin (AM) in mammals. In teleost fish, AM forms an independent subfamily of five members (AM1–5), which inspired us to trace the evolutionary history of the CGRP family throughout vertebrates by comparative genomic approach. Linkage mapping and synteny analyses of the CGRP family genes in medaka, Oryzias latipes, revealed that AM1/CGRP, AM2/amylin, and AM5 genes were located on respective proto-chromosomes before the divergence of teleost lineage. In teleost fish, additional whole genome duplication generated AM1/4, CGRP1/2, AM2/3, but one of the duplicated amylin and AM5 genes was silenced. In mammals, the amylin or AM2 gene was translocated to different chromosomes, while the CGRP gene was multiplied in tandem to generate CGRP-,β, and recently identified calcitonin receptor-stimulating peptide genes. Based on these data, we identified a novel AM5 gene in several mammalian species as we previously did for AM2.     

Common ancestry and novel genetic traits of Francisella novicida-like isolates from North America and Australia as revealed by comparative genomic analyses

Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.     

Gene-associated CpG islands in plants as revealed by analyses of genomic sequences

We screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG-rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG-rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG-rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5'-end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5'-end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue-specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5'-end of the genes.     

Organ specificity and transcriptional control of metabolic routes revealed by expression QTL profiling of source-sink tissues in a segregating potato population

Background  

With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization.     

Functions of mammalian Smad genes as revealed by targeted gene disruption in mice

The Smad genes are the intracellular mediators of TGF-beta signals. Targeted mutagenesis in mice has yielded valuable new insights into the functions of this important gene family. These experiments have shown that Smad2 and Smad4 are needed for gastrulation, Smad5 for angiogenesis, and Smad3 for establishment of the mucosal immune response and proper development of the skeleton. In addition, these experiments have shown us the importance of gene dosage in this family, as several of its members yielded haploinsufficiency phenotypes. These include gastrulation and craniofacial defects for Smad2, accelerated wound healing for Smad3, and the incidence of gastric cancer for Smad4. Combinatorial genetics has also revealed functions of Smads in left/right isomerism and liver development.     

Alteration of raw-milk cheese by Pseudomonas spp.: monitoring the sources of contamination using fluorescence spectroscopy and metabolic profiling

Thirty Pseudomonas spp. strains isolated from milk, water, cheese centre and cheese surface in two traditional workshops manufacturing raw milk St. Nectaire cheese were characterised by fluorescence spectroscopy and Biolog metabolic profiling. Factorial discriminant analysis (FDA) of the two data sets revealed clear linkages between groups of isolates. In the first workshop, milk could be incriminated as the sole source of cheese contamination. In the second one, milk and cheese centre isolates were found similar but surface cheese contaminants could be linked to a secondary contamination originating from water. Thus, it is possible to characterise, differentiate and trace Pseudomonas spp. strains using the fluorescence and metabolic profiling techniques. In addition, the two data sets were found highly correlated by canonical correlation analysis (CCA). Fluorescence spectroscopy however showed several advantages because of its low cost and processing speed.