Mitochondrial misreading in skeletal muscle accelerates metabolic aging and confers lipid accumulation and increased inflammation.

We have recently reported on an experimental model of mitochondrial mistranslation conferred by amino acid exchange V338Y in the mitochondrial ribosomal protein MrpS5. Here we used a combination of RNA-Seq and metabolic profiling of homozygous transgenic MrpS5V338Y/V338Y mice to analyze the changes associated with the V338Y mutation in post-mitotic skeletal muscle. Metabolic profiling demonstrated age-dependent metabolic changes in the mutant V338Y animals, which included enhanced levels of age-associated metabolites and which were accompanied by increased glycolysis, lipid desaturation and eicosanoid biosynthesis, and alterations of the pentose phosphate pathway. In addition, transcriptome signatures of aged V338Y mutant muscle pointed to elevated inflammation, likely reflecting the increased levels of bioactive lipids. Our findings indicate that mistranslation-mediated chronic impairment of mitochondrial function affects specific bioenergetic processes in muscle in an age-dependent manner.

Phenotype of Mrps5-Associated Phylogenetic Polymorphisms Is Intimately Linked to Mitoribosomal Misreading.

We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.

Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle.

Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of ß-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

Metabolomic Profiling and Toxicokinetics Modeling to Assess the Effects of the Pharmaceutical Diclofenac in the Aquatic Invertebrate Hyalella azteca.

The exposure of ecologically critical invertebrate species to biologically active pharmaceuticals poses a serious risk to the aquatic ecosystem. Yet, the fate and toxic effects of pharmaceuticals on these nontarget aquatic invertebrates and the underlying mechanisms are poorly studied. Herein, we investigated the toxicokinetic (TK) processes (i.e., uptake, biotransformation, and elimination) of the pharmaceutical diclofenac and its biotransformation in the freshwater invertebrate Hyalella azteca. We further employed mass spectrometry-based metabolomics to assess the toxic effects of diclofenac on the metabolic functions of H. azteca exposed to environmentally relevant concentrations (10 and 100 µg/L). The TK results showed a quick uptake of diclofenac by H. azteca (maximum internal concentration of 1.9 µmol/kg) and rapid formation of the conjugate diclofenac taurine (maximum internal concentration of 80.6 µmol/kg), indicating over 40 times higher accumulation of diclofenac taurine than that of diclofenac in H. azteca. Depuration kinetics demonstrated that the elimination of diclofenac taurine was 64 times slower than diclofenac in H. azteca. Metabolomics results suggested that diclofenac inhibited prostaglandin synthesis and affected the carnitine shuttle pathway at environmentally relevant concentrations. These findings shed light on the significance of the TK process of diclofenac, especially the formation of diclofenac taurine, as well as the sublethal effects of diclofenac on the bulk metabolome of H. azteca. Combining the TK processes and metabolomics provides complementary insights and thus a better mechanistic understanding of the effects of diclofenac in aquatic invertebrates.

Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression.

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

Analysis of the equine "cumulome" reveals major metabolic aberrations after maturation in vitro.

BACKGROUND: Maturation of oocytes under in vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in vivo. As oocytes are closely coupled to their cumulus complex, elucidating aberrations in cumulus metabolism in vitro is important to bridge the gap towards more physiological maturation conditions. The aim of this study was to analyze the equine "cumulome" in a novel combination of proteomic (nano-HPLC MS/MS) and metabolomic (UPLC-nanoESI-MS) profiling of single cumulus complexes of metaphase II oocytes matured either in vivo (n = 8) or in vitro (n = 7). RESULTS: A total of 1811 quantifiable proteins and 906 metabolic compounds were identified. The proteome contained 216 differentially expressed proteins (p ≤ 0.05; FC ≥ 2; 95 decreased and 121 increased in vitro), and the metabolome contained 108 metabolites with significantly different abundance (p ≤ 0.05; FC ≥ 2; 24 decreased and 84 increased in vitro). The in vitro "cumulome" was summarized in the following 10 metabolic groups (containing 78 proteins and 21 metabolites): (1) oxygen supply, (2) glucose metabolism, (3) fatty acid metabolism, (4) oxidative phosphorylation, (5) amino acid metabolism, (6) purine and pyrimidine metabolism, (7) steroid metabolism, (8) extracellular matrix, (9) complement cascade and (10) coagulation cascade. The KEGG pathway "complement and coagulation cascades" (ID4610; n = 21) was significantly overrepresented after in vitro maturation. The findings indicate that the in vitro condition especially affects central metabolism and extracellular matrix composition. Important candidates for the metabolic group oxygen supply were underrepresented after maturation in vitro. Additionally, a shift towards glycolysis was detected in glucose metabolism. Therefore, under in vitro conditions, cumulus cells seem to preferentially consume excess available glucose to meet their energy requirements. Proteins involved in biosynthetic processes for fatty acids, cholesterol, amino acids, and purines exhibited higher abundances after maturation in vitro. CONCLUSION: This study revealed the marked impact of maturation conditions on the "cumulome" of individual cumulus oocyte complexes. Under the studied in vitro milieu, cumulus cells seem to compensate for a lack of important substrates by shifting to aerobic glycolysis. These findings will help to adapt culture media towards more physiological conditions for oocyte maturation.

Sorbitol dehydrogenase overexpression and other aspects of dysregulated protein expression in human precancerous colorectal neoplasms: a quantitative proteomics study.

Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated via OFFGEL electrophoresis, and analyzed by means of LC-MS/MS. Nonredundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples and cancer cell lines from HCEC cells. Two hundred and twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change versus normal mucosa ±1.4), which correlated (r = 0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (∼25%) proteins displayed directionally similar expression changes in colorectal cancer cells (versus HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein up-regulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase, a key enzyme in the polyol pathway. Validation studies revealed dramatically increased sorbitol dehydrogenase concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism might represent a novel facet of metabolome remodeling associated with tumorigenesis.

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter.

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.

Identification of Iodotyrosines as Novel Substrates for the Thyroid Hormone Transporter MCT8.

Background: Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe intellectual and motor disability and abnormal serum thyroid function tests. However, it is presently unknown if MCT8, similar to other thyroid hormone transporters, also accepts additional substrates, and if disruption of their transport may contribute to the observed phenotype. Methods: In this study, we aimed to identify such substrates by applying liquid chromatography-mass spectrometry-based metabolome analysis in lysates of control and MCT8-overexpressing Xenopus oocytes. A subset of identified candidate substrates were validated by direct transport studies in transiently transfected COS-1 cells and human fibroblasts, which endogenously express MCT8. Moreover, transport characteristics were determined, including transport saturation and cis-inhibition potency of thyroid hormone transport. Results: Metabolome analysis identified 21 m/z ratios, corresponding to 87 candidate metabolites, with a 2.0-times differential abundance in MCT8-injected oocytes compared with controls. These metabolites included 3,5-diiodotyrosine (DIT) and several amino acids, including glutamate and glutamine. In accordance, MCT8-expressing COS-1 cells had 2.2-times lower intracellular accumulation of [125I]-DIT compared with control cells. This effect was largely blocked in the presence of 3,3',5-triiodothyronine (T3) (IC50: 2.5 ± 1.5 µM) or thyroxine (T4) (IC50: 5.8 ± 1.3 µM). Conversely, increasing concentrations of DIT enhanced the accumulation of T3 and T4. The MCT8-specific inhibitor silychristin increased the intracellular accumulation of DIT in human fibroblasts. COS-1 cells expressing MCT8 also exhibited a 50% reduction in intracellular accumulation of [125I]-3-monoiodotyrosine (MIT). In contrast, COS-1 cells expressing MCT8 did not alter the intracellular accumulation of [3H]-glutamate or [3H]-glutamine. However, studies in human fibroblasts showed a 1.5-1.9 times higher glutamate uptake in control fibroblasts compared with fibroblasts derived from patients with MCT8 deficiency, which was not affected in the presence of silychristin. Conclusions: Taken together, our results suggest that the iodotyrosines DIT and MIT can be exported by MCT8. MIT and DIT interfere with MCT8-mediated transport of thyroid hormone in vitro and vice versa. Future studies should elucidate if MCT8, being highly expressed in thyroidal follicular cells, also transports iodotyrosines in vivo.

Multiomic ALS signatures highlight subclusters and sex differences suggesting the MAPK pathway as therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.

Serotonin protects mouse liver from cholestatic injury by decreasing bile salt pool after bile duct ligation.

UNLABELLED: Obstructive cholestasis induces liver injury, postoperative complications, and mortality after surgery. Adaptive control of cholestasis, including bile salt homeostasis, is necessary for recovery and survival. Peripheral serotonin is a cytoprotective neurotransmitter also associated with liver regeneration. The effect of serotonin on cholestatic liver injury is not known. Therefore, we tested whether serotonin affects the severity of cholestatic liver injury. We induced cholestasis by ligation of the bile duct (BDL) in either wild-type (WT) mice or mice lacking peripheral serotonin (Tph1(-/-) and immune thrombocytopenic [ITP] mice). Liver injury was assessed by the levels of plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and tissue necrosis. Bile salt-regulating genes were measured by quantitative polymerase chain reaction and confirmed by western blotting and immunohistochemistry. Tph1(-/-) mice displayed higher levels of plasma AST, ALT, bile salts, and hepatic necrosis after 3 days of BDL than WT mice. Likewise, liver injury was disproportional in ITP mice. Moreover, severe cholestatic complications and mortality after prolonged BDL were increased in Tph1(-/-) mice. Despite the elevation in toxic bile salts, expression of genes involved in bile salt homeostasis and detoxification were not affected in Tph1(-/-) livers. In contrast, the bile salt reabsorption transporters Ostα and Ostß were up-regulated in the kidneys of Tph1(-/-) mice, along with a decrease in urinary bile salt excretion. Serotonin reloading of Tph1(-/-) mice reversed this phenotype, resulting in a reduction of circulating bile salts and liver injury. CONCLUSION: We propose a physiological function of serotonin is to ameliorate liver injury and stabilize the bile salt pool through adaptation of renal transporters in cholestasis.

A Metabolome Analysis and the Immunity of Phlomis purpurea against Phytophthora cinnamomi.

Phlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens' biological control. Its application in green forestry/agriculture is therefore possible.

Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization.

Differentiation is critical for cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in preclinical models of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.

Phosphoproteome profile of Fusarium graminearum grown in vitro under nonlimiting conditions.

This study presents a high-throughput proteomic analysis of phosphopeptides from Fusarium graminearum strain DAOM 233423 grown in vitro without nutritional limitation. Using a combination of strong cation exchange (SCX) and immobilized metal affinity chromatography (IMAC) followed by LC-MS, we identified 2902 putative phosphopeptides with homologous matches to 1496 different proteins. Functional classification of the annotated protein set revealed that phosphopeptides from nuclear proteins with ATP-binding function were the most abundant. There are indications that phosphorylation sites from well-characterized phosphoproteins representing diverse biological processes are conserved in F. graminearum: sequences of three phosphopeptides from known phosphoproteins (transcription elongation factor 1ß, acidic ribosomal proteins, and glycogen synthase) revealed phosphorylation site conservation.

Molecular pathways undergoing dramatic transcriptomic changes during tumor development in the human colon.

BACKGROUND: The malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations. METHODS: We used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa). RESULTS: Of the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics). CONCLUSIONS: Our analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.

Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster.

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.

Association of proteomic markers with nutritional risk and response to nutritional support: A secondary pilot study of the EFFORT trial using an untargeted proteomics approach.

BACKGROUND: By means of a structured nutritional support intervention, EFFORT showed a risk reduction for adverse events in medical in-patients. We were interested in the prognostic and therapeutic potential of an untargeted proteomics approach to understand response to nutritional support, risk of 30-day mortality, and distinct patterns in severity of malnutrition risk as assessed by the Nutritional Risk screening (NRS 2002), respectively. METHODS: From 2,088 patients, we randomly took 120 blood samples drawn before treatment initiation on day 1 after hospital admission. Cases were selected by treatment allocation (nutritional support vs. usual nutrition), NRS 2002, and mortality at 30 days, but not on disease type. We measured proteins by untargeted liquid chromatography mass spectrometry (LC-MS/MS). RESULTS: We found 242 distinct proteins in 120 patients of which 81 (67.5%) survived until day 30. Between group analysis revealed a slight difference between the treatment groups in patients with a NRS 3, but not in those with a higher NRS. C-statistic between non-survivors and survivors at day 30 ranged from 0.60 (95% confidence interval 0.34-0.78) for a combination of 3 proteins/predictors to 0.65 (95% CI 0.53-0.78) for a combination of 32 proteins/predictors. In nutritional support non-survivors, pathway analysis found significant enrichment in pathways for signal transduction, platelet function, immune system regulation, extracellular matrix organization, and integrin cell surface interactions compared to survivors. CONCLUSION: Within this pilot study using an untargeted proteomics approach, there was only little prognostic and therapeutic potential of proteomics for phenotyping the risk of malnutrition and response to nutritional therapy. The small sample size and high heterogeneity of our population regarding comorbidity burden calls for more targeted approaches in more homogenous populations to understand the true potential of proteomics for individualizing nutritional care. TRIAL REGISTRATION: This is a pre-planned secondary analysis of the EFFORT trial (ClinicalTrials.gov NCT02517476).

Chemical composition of hepatic lipids mediates reperfusion injury of the macrosteatotic mouse liver through thromboxane A(2).

BACKGROUND & AIMS: Chemical composition of hepatic lipids is an evolving player in steatotic liver ischemia/reperfusion (I/R) injury. Thromboxane A(2) (TXA(2)) is a vasoactive pro-inflammatory lipid mediator derived from arachidonic acid (AA), an omega-6 fatty acid (Ω-6 FA). Reduced tolerance of the macrosteatotic liver to I/R may be related to increased TXA(2) synthesis due to the predominance of Ω-6 FAs. METHODS: TXA(2) levels elicited by I/R in ob/ob and wild type mice were assessed by ELISA. Ob/ob mice were fed Ω-3 FAs enriched diet to reduce hepatic synthesis of AA and TXA(2) or treated with selective TXA(2) receptor blocker before I/R. RESULTS: I/R triggered significantly higher hepatic TXA(2) production in ob/ob than wild type animals. Compared with ob/ob mice on regular diet, Ω-3 FAs supplementation markedly reduced hepatic AA levels before ischemia and consistently blunted hepatic TXA(2) synthesis after reperfusion. Sinusoidal perfusion and hepatocellular damage were significantly ameliorated despite downregulation of heme oxygenase-1. Hepatic transcript and protein levels of IL-1ß and neutrophil recruitment were significantly diminished after reperfusion. Moreover, TXA(2) receptor blockage conferred similar protection without modification of the histological pattern of steatosis. A stronger protection was achieved in the steatotic compared with lean animals. CONCLUSIONS: Enhanced I/R injury in the macrosteatotic liver is explained, at least partially, by TXA(2) mediated microcirculatory failure rather than size-related mechanical compression of the sinusoids by lipid droplets. TXA(2) blockage may be a simple strategy to include steatotic organs and overcome the shortage of donor organs for liver transplantation.

Fibrella aestuarina gen. nov., sp. nov., a filamentous bacterium of the family Cytophagaceae isolated from a tidal flat, and emended description of the genus Rudanella Weon et al. 2008.

A Gram-staining-negative, pink bacterium, designated strain BUZ 2(T), was isolated from coastal mud from the North Sea (Fedderwardersiel, Germany). Cells were rod-shaped and able to form multicellular filaments. Growth after 7 days was observed at 10-40 °C, at pH 6-8 and with 0-0.5 % NaCl. The phylogenetic tree based on 16S rRNA gene sequences indicated that strain BUZ 2(T) is a member of the family Cytophagaceae, its closest neighbours being Rudanella lutea 5715S-11(T), Spirosoma linguale LMG 10896(T) and Spirosoma panaciterrae Gsoil 1519(T) (87.8, 86.4 and 86.1 % sequence similarity, respectively). The major fatty acids were summed feature 3 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH), C(16 : 1)ω5c and iso-C(15 : 0). The predominant respiratory quinone was MK-7 and the major polar lipids were phosphatidylethanolamine and several unidentified aminophospholipids. The DNA G+C content was 56.5 mol%. On the basis of this polyphasic study, we propose that strain BUZ 2(T) represents a novel genus and species, for which the name Fibrella aestuarina gen. nov., sp. nov. is proposed. The type strain of Fibrella aestuarina is BUZ 2(T) (=DSM 22563(T) =CCUG 58136(T)). An emended description of the genus Rudanella is also proposed.

Association of metabolomic markers and response to nutritional support: A secondary analysis of the EFFORT trial using an untargeted metabolomics approach.

BACKGROUND & AIMS: The EFFORT trial reported a substantial risk reduction for adverse events and mortality in medical in-patients receiving a nutritional support intervention. With the use of an untargeted metabolomics approach, we investigated the prognostic and therapeutic potential of metabolomic markers to understand, whether there are distinct metabolic patterns associated with malnutrition risk as assessed by the Nutritional Risk screening (NRS 2002) score, the risk of 30-day mortality and the response to nutritional support, respectively. METHODS: Out of the 2088 samples we randomly selected 120 blood samples drawn on day 1 after hospital admission and before treatment initiation. Samples were stratified by NRS 2002, treatment allocation (intervention vs. control), and mortality at 30 days, but not on the type of medical illness. We performed untargeted analysis by liquid chromatography mass spectrometry (LC-MS/MS). RESULTS: We measured 1389 metabolites in 120 patients of which 81 (67.5%) survived until day 30. After filtering, 371 metabolites remained, and 200 were matched to one or more Human Metabolome Data Base (HMDB) entries. Between group analysis showed a slight distinction between the treatment groups for patients with a NRS 3, but not for those with NRS 4 and ≥ 5. C-statistic between those who died and survived at day 30 ranged from 0.49 (95% confidence interval 0.35-0.68) for a combination of 5 metabolites/predictors to 0.66 (95% confidence interval 0.53-0.79) for a combination of 100 metabolites. Pathway analysis found significant enrichment in the pathways for nitrogen, vitamin B3 (nicotinate and nicotinamide), leukotriene, and arachidonic acid metabolisms in nutritional support responders compared to non-responders. CONCLUSION: In our heterogenous population of medical inpatients with different illnesses and comorbidities, metabolomic markers showed little prognostic and therapeutic potential for better phenotyping malnutrition and response to nutritional therapy. Future studies should focus on more selected patient populations to understand whether a metabolomic approach can advance the nutritional care of patients.