Multiligament knee injuries. Ten years' experience at a public university, level I Trauma Center.

PURPOSE: To describe our institutional experience and results in the surgical management of multiligament knee injuries (MLKI). MATERIALS AND METHODS: Retrospective series of MLKI consecutively operated on at a single, level I Trauma Center. Data on patients' baseline characteristics, injuries, treatments, and outcomes were recorded up to one-year follow-up. Recorded outcomes included the Tegner-Lysholm Knee Scoring Scale (TLKSS), return to work, and patient satisfaction. RESULTS: MLKI incidence was 0.03% among 9897 orthopedic trauma admissions. Twenty-four patients of mean age 43.6 years were included in analysis. The mean Injury Severity Score was 12.6. Five patients presented with knee dislocations and six had fracture-dislocations, two of them open fractures. There was one popliteal artery injury requiring a bypass and four common peroneal nerve palsies. Staged ligamental reconstruction was performed in all cases. There were seven postoperative complications. The median TLKSS was 80 and, though patient satisfaction was high, and dissatisfaction was largely restricted to recreational activities (only 58.3% satisfied). Seventeen patients returned to their previous employment. CONCLUSIONS: We found a high aggregation of fracture-dislocations secondary to road traffic accidents. One in four patients experienced complications, particularly stiffness. Complications were more common in cases involving knee dislocation. Most patients had good functional results, but 25% were unable to return to their previous work, which demonstrates the long-lasting sequelae of this injury.

Good rates of return-to-sport in athletes after revision anterior cruciate ligament reconstruction using autologous patellar tendon and lateral extra-articular tenodesis: a 2-year follow-up prospective study.

BACKGROUND: Most athletes who undergo revision of the anterior cruciate ligament reconstruction (ACLR) aim to return to their preinjury sport at a similar level of performance while minimizing the risk for reinjury. Additional lateral extra-articular tenodesis (LET) has recently been correlated with improved outcomes and low complication rate. Yet, there are few series evaluating return-to-sport (RTS) and clinical outcomes after revision ACLR using bone-patellar tendon-bone (BPTB) and LET in athletes. METHODS: The study cohort consisted of 19 eligible athletes who had undergone their first revision ACLR using BPTB and LET (modified Lemaire) between January 2019 and 2020. Patients were prospectively followed and interviewed in a sports activity survey during a 2-year follow-up. RESULTS: Despite all patients returning to sports after revision ACLR surgery, 52.6% resumed playing at their preinjury level. Furthermore, patient-reported functional outcomes improved significantly following revision surgery, as evidenced by improvements in IKDC [64.4 (± 12) to 87.8 (± 6)], Lysholm [71.27 (± 12) to 84.2 (± 9.7)], and SF-12 scales [Physical: 53.3 (± 3) 57 (± 1.2); Mental: 50.2 (± 3.3) to 52.7 (± 2.4)]. One case (5.3%) experienced persistent pain and underwent reoperation for a partial meniscectomy. CONCLUSION: After revision ACLR using autologous BPTB and LET, all active individuals are expected to RTS, similar to primary ACLR. The difference comes down to returning to the preinjury level, where the levels are lower depending on the sport and initial level of play. Good mid-term functional outcomes with a low complication rate can be expected in most cases. STUDY DESIGN: Case series; Level of evidence IV. ETHICAL COMMITTEE APPROVAL NUMBER: PR(ATR)79/2021 and HCB/2023/0173.

Metabolic reprogramming by Acly inhibition using SB-204990 alters glucoregulation and modulates molecular mechanisms associated with aging.

ATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically induced Acly inhibition are unknown. We report here that the Acly inhibitor SB-204990 improves metabolic health and physical strength in wild-type mice when fed with a high-fat diet, while in mice fed with healthy diet results in metabolic imbalance and moderated insulin resistance. By applying a multiomic approach using untargeted metabolomics, transcriptomics, and proteomics, we determined that, in vivo, SB-204990 plays a role in the regulation of molecular mechanisms associated with aging, such as energy metabolism, mitochondrial function, mTOR signaling, and folate cycle, while global alterations on histone acetylation are absent. Our findings indicate a mechanism for regulating molecular pathways of aging that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be explored for devising therapeutic approaches to prevent metabolic diseases.

Allografts and lateral extra-articular tenodesis for revision anterior cruciate ligament reconstruction: enhanced rotational stability and improved functional outcomes.

PURPOSE: Multiple studies have shown higher failure rate and patient-reported outcomes to be significantly worse following revision anterior cruciate ligament reconstructive (ACLR) surgery, especially using allografts. One of the reasons being rotational instability. Because of this, augmentation with lateral extra-articular tenodesis (LET) is often considered. Good short-term results in regards to functional and perceived scores and low complication rate can be expected in revision ACLR using allografts in combination with LET. METHODS: Between 2014 and 2021, 46 patients were registered for revision ACLR using allografts and extra-articular augmentation (modified Lemaire) and included in this prospective study. Patients' demographic and clinical data were collected preoperatively, postoperatively, and during the follow-up period of 12 months. RESULTS: Patient-reported functional outcomes were statistically significant for IKDC, Lysholm, and SF-12 physical scale (p < 0.05). Tegner score showed a decreased number of patients who were able to return to sport at their previous level (p = 0.001). Stability examination tests (Lachman and pivot-shift) showed significant improvements. Concomitant lesions were present in 76.1% of patients. Ten patients (21.7%) presented major complications, including six cases of anteroposterior instability, three cases of knee pain and one graft re-rupture. CONCLUSION: Revision procedures are inherently challenging with a high number of associated chondral and meniscus lesions. However, good short-term functional outcomes and enhanced rotational stability with an acceptable complication rate can be expected in most cases where revision ACLR using allografts is augmented with LET. STUDY DESIGN: Prospective; Case series; Level of evidence IV.

Muscular carnosine is a marker for cardiorespiratory fitness and cardiometabolic risk factors in men with type 1 diabetes.

PURPOSE: Muscle is an essential organ for glucose metabolism and can be influenced by metabolic disorders and physical activity. Elevated muscle carnosine levels have been associated with insulin resistance and cardiometabolic risk factors. Little is known about muscle carnosine in type 1 diabetes (T1D) and how it is influenced by physical activity. The aim of this study was to characterize muscle carnosine in vivo by proton magnetic resonance spectroscopy (1H MRS) and evaluate the relationship with physical activity, clinical characteristics and lipoprotein subfractions. METHODS: 16 men with T1D (10 athletes/6 sedentary) and 14 controls without diabetes (9/5) were included. Body composition by DXA, cardiorespiratory capacity (VO2peak) and serum lipoprotein profile by proton nuclear magnetic resonance (1H NMR) were obtained. Muscle carnosine scaled to water (carnosineW) and to creatine (carnosineCR), creatine and intramyocellular lipids (IMCL) were quantified in vivo using 1H MRS in a 3T MR scanner in soleus muscle. RESULTS: Subjects with T1D presented higher carnosine CR levels compared to controls. T1D patients with a lower VO2peak presented higher carnosineCR levels compared to sedentary controls, but both T1D and control groups presented similar levels of carnosineCR at high VO2peak levels. CarnosineW followed the same trend. Integrated correlation networks in T1D demonstrated that carnosineW and carnosineCR were associated with cardiometabolic risk factors including total and abdominal fat, pro-atherogenic lipoproteins (very low-density lipoprotein subfractions), low VO2peak, and IMCL. CONCLUSIONS: Elevated muscle carnosine levels in persons with T1D and their effect on atherogenic lipoproteins can be modulated by physical activity.

Toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in early development: A wide-scope metabolomics assay in zebrafish embryos.

The tobacco-specific nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic and ubiquitous environmental pollutant for which toxic activity has been thoroughly investigated in murine models and human tissues. However, its potential deleterious effects on vertebrate early development are yet poorly understood. In this work, we characterized the impact of NNK exposure during early developmental stages of zebrafish embryos, a known alternative model for mammalian toxicity studies. Embryos exposed to different NNK concentrations were monitored for lethality and for the appearance of malformations during the first five days after fertilization. LC-MS based untargeted metabolomics was subsequently performed for a wide-scope assay of NNK-related metabolic alterations. Our results revealed the presence of not only the parental compound, but also of two known NNK metabolites, 4-Hydroxy-4-(3-pyridyl)-butyric acid (HPBA) and 4-(Methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol (NNAL-N-oxide) in exposed embryos likely resulting from active CYP450-mediated α-hydroxylation and NNK detoxification pathways, respectively. This was paralleled by a disruption in purine and pyrimidine metabolisms and the activation of the base excision repair pathway. Our results confirm NNK as a harmful embryonic agent and demonstrate zebrafish embryos to be a suitable early development model to monitor NNK toxicity.

HERMES: a molecular-formula-oriented method to target the metabolome.

Comprehensive metabolome analyses are essential for biomedical, environmental, and biotechnological research. However, current MS1- and MS2-based acquisition and data analysis strategies in untargeted metabolomics result in low identification rates of metabolites. Here we present HERMES, a molecular-formula-oriented and peak-detection-free method that uses raw LC/MS1 information to optimize MS2 acquisition. Investigating environmental water, Escherichia coli, and human plasma extracts with HERMES, we achieved an increased biological specificity of MS2 scans, leading to improved mass spectral similarity scoring and identification rates when compared with a state-of-the-art data-dependent acquisition (DDA) approach. Thus, HERMES improves sensitivity, selectivity, and annotation of metabolites. HERMES is available as an R package with a user-friendly graphical interface for data analysis and visualization.

Increased Hypothalamic Anti-Inflammatory Mediators in Non-Diabetic Insulin Receptor Substrate 2-Deficient Mice.

Insulin receptor substrate (IRS) 2 is a key mediator of insulin signaling and IRS-2 knockout (IRS2-/-) mice are a preclinical model to study the development of diabetes, as they develop peripheral insulin resistance and beta-cell failure. The differential inflammatory profile and insulin signaling in the hypothalamus of non-diabetic (ND) and diabetic (D) IRS2-/- mice might be implicated in the onset of diabetes. Because the lipid profile is related to changes in inflammation and insulin sensitivity, we analyzed whether ND IRS2-/- mice presented a different hypothalamic fatty acid metabolism and lipid pattern than D IRS2-/- mice and the relationship with inflammation and markers of insulin sensitivity. ND IRS2-/- mice showed elevated hypothalamic anti-inflammatory cytokines, while D IRS2-/- mice displayed a proinflammatory profile. The increased activity of enzymes related to the pentose-phosphate route and lipid anabolism and elevated polyunsaturated fatty acid levels were found in the hypothalamus of ND IRS2-/- mice. Conversely, D IRS2-/- mice have no changes in fatty acid composition, but hypothalamic energy balance and markers related to anti-inflammatory and insulin-sensitizing properties were reduced. The data suggest that the concurrence of an anti-inflammatory profile, increased insulin sensitivity and polyunsaturated fatty acids content in the hypothalamus may slow down or delay the onset of diabetes.

Untargeted lipidomics uncovers lipid signatures that distinguish severe from moderate forms of acutely decompensated cirrhosis.

BACKGROUND & AIMS: Acute decompensation (AD) of cirrhosis is a heterogeneous clinical entity associated with moderate mortality. In some patients, this condition develops quickly into the more deadly acute-on-chronic liver failure (ACLF), in which other organs such as the kidneys or brain fail. The aim of this study was to characterize the blood lipidome in a large series of patients with cirrhosis and identify specific signatures associated with AD and ACLF development. METHODS: Serum untargeted lipidomics was performed in 561 patients with AD (518 without and 43 with ACLF) (discovery cohort) and in 265 patients with AD (128 without and 137 with ACLF) in whom serum samples were available to perform repeated measurements during the 28-day follow-up (validation cohort). Analyses were also performed in 78 patients with AD included in a therapeutic albumin trial (43 patients with compensated cirrhosis and 29 healthy individuals). RESULTS: The circulating lipid landscape associated with cirrhosis was characterized by a generalized suppression, which was more manifest during AD and in non-surviving patients. By computing discriminating accuracy and the variable importance projection score for each of the 223 annotated lipids, we identified a sphingomyelin fingerprint specific for AD of cirrhosis and a distinct cholesteryl ester and lysophosphatidylcholine fingerprint for ACLF. Liver dysfunction and infections were the principal net contributors to these fingerprints, which were dynamic and interchangeable between patients with AD whose condition worsened to ACLF and those who improved. Notably, blood lysophosphatidylcholine levels increased in these patients after albumin therapy. CONCLUSIONS: Our findings provide insights into the lipid landscape associated with decompensation of cirrhosis and ACLF progression and identify unique non-invasive diagnostic biomarkers of advanced cirrhosis. LAY SUMMARY: Analysis of lipids in blood from patients with advanced cirrhosis reveals a general suppression of their levels in the circulation of these patients. A specific group of lipids known as sphingomyelins are useful to distinguish between patients with compensated and decompensated cirrhosis. Another group of lipids designated cholesteryl esters further distinguishes patients with decompensated cirrhosis who are at risk of developing organ failures.

Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics.

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC's superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.

Integrated Probabilistic Annotation: A Bayesian-Based Annotation Method for Metabolomic Profiles Integrating Biochemical Connections, Isotope Patterns, and Adduct Relationships.

In a typical untargeted metabolomics experiment, the huge amount of complex data generated by mass spectrometry necessitates automated tools for the extraction of useful biological information. Each metabolite generates numerous mass spectrometry features. The association of these experimental features to the underlying metabolites still represents one of the major bottlenecks in metabolomics data processing. While certain identification (e.g., by comparison to authentic standards) is always desirable, it is usually achievable only for a limited number of compounds, and scientists often deal with a significant amount of putatively annotated metabolites. The confidence in a specific annotation is usually assessed by considering different sources of information (e.g., isotope patterns, adduct formation, chromatographic retention times, and fragmentation patterns). IPA (integrated probabilistic annotation) offers a rigorous and reproducible method to automatically annotate metabolite profiles and evaluate the resulting confidence of the putative annotations. It is able to provide a rigorous measure of our confidence in any putative annotation and is also able to update and refine our beliefs (i.e., background prior knowledge) by incorporating different sources of information in the annotation process, such as isotope patterns, adduct formation and biochemical relations. The IPA package is freely available on GitHub ( https://github.com/francescodc87/IPA ), together with the related extensive documentation.

Machine Learning of Designed Translational Control Allows Predictive Pathway Optimization in Escherichia coli.

The field of synthetic biology aims to make the design of biological systems predictable, shrinking the huge design space to practical numbers for testing. When designing microbial cell factories, most optimization efforts have focused on enzyme and strain selection/engineering, pathway regulation, and process development. In silico tools for the predictive design of bacterial ribosome binding sites (RBSs) and RBS libraries now allow translational tuning of biochemical pathways; however, methods for predicting optimal RBS combinations in multigene pathways are desirable. Here we present the implementation of machine learning algorithms to model the RBS sequence-phenotype relationship from representative subsets of large combinatorial RBS libraries allowing the accurate prediction of optimal high-producers. Applied to a recombinant monoterpenoid production pathway in Escherichia coli, our approach was able to boost production titers by over 60% when screening under 3% of a library. To facilitate library screening, a multiwell plate fermentation procedure was developed, allowing increased screening throughput with sufficient resolution to discriminate between high and low producers. High producers from one library did not translate during scale-up, but the reduced screening requirements allowed rapid rescreening at the larger scale. This methodology is potentially compatible with any biochemical pathway and provides a powerful tool toward predictive design of bacterial production chassis.

FELLA: an R package to enrich metabolomics data.

BACKGROUND: Pathway enrichment techniques are useful for understanding experimental metabolomics data. Their purpose is to give context to the affected metabolites in terms of the prior knowledge contained in metabolic pathways. However, the interpretation of a prioritized pathway list is still challenging, as pathways show overlap and cross talk effects. RESULTS: We introduce FELLA, an R package to perform a network-based enrichment of a list of affected metabolites. FELLA builds a hierarchical representation of an organism biochemistry from the Kyoto Encyclopedia of Genes and Genomes (KEGG), containing pathways, modules, enzymes, reactions and metabolites. In addition to providing a list of pathways, FELLA reports intermediate entities (modules, enzymes, reactions) that link the input metabolites to them. This sheds light on pathway cross talk and potential enzymes or metabolites as targets for the condition under study. FELLA has been applied to six public datasets -three from Homo sapiens, two from Danio rerio and one from Mus musculus- and has reproduced findings from the original studies and from independent literature. CONCLUSIONS: The R package FELLA offers an innovative enrichment concept starting from a list of metabolites, based on a knowledge graph representation of the KEGG database that focuses on interpretability. Besides reporting a list of pathways, FELLA suggests intermediate entities that are of interest per se. Its usefulness has been shown at several molecular levels on six public datasets, including human and animal models. The user can run the enrichment analysis through a simple interactive graphical interface or programmatically. FELLA is publicly available in Bioconductor under the GPL-3 license.

Metabolomic Response to Acute Hypoxic Exercise and Recovery in Adult Males.

Metabolomics is a relatively new "omics" approach used to characterize metabolites in a biological system at baseline and following a diversity of stimuli. However, the metabolomic response to exercise in hypoxia currently remains unknown. To examine this, 24 male participants completed 1 h of exercise at a workload corresponding to 75% of pre-determined O2max in hypoxia (Fio2 = 0.16%), and repeated in normoxia (Fio2 = 0.21%), while pre- and post-exercise and 3 h post-exercise metabolites were analyzed using a LC ESI-qTOF-MS untargeted metabolomics approach in serum samples. Exercise in hypoxia and in normoxia independently increased metabolism as shown by a change in a combination of twenty-two metabolites associated with lipid metabolism (p < 0.05, pre vs. post-exercise), though hypoxia per se did not induce a greater metabolic change when compared with normoxia (p > 0.05). Recovery from exercise in hypoxia independently decreased seventeen metabolites associated with lipid metabolism (p < 0.05, post vs. 3 h post-exercise), compared with twenty-two metabolites in normoxia (p < 0.05, post vs. 3 h post-exercise). Twenty-six metabolites were identified as responders to exercise and recovery (pooled hypoxia and normoxia pre vs. recovery, p < 0.05), including metabolites associated with purine metabolism (adenine, adenosine and hypoxanthine), the amino acid phenylalanine, and several acylcarnitine molecules. Our novel data provides preliminary evidence of subtle metabolic differences to exercise and recovery in hypoxia and normoxia. Specifically, exercise in hypoxia activates metabolic pathways aligned to purine and lipid metabolism, but this effect is not selectively different from exercise in normoxia. We also show that exercise per se can activate pathways associated with lipid, protein and purine nucleotide metabolism.

An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals.

The microbial production of fine chemicals provides a promising biosustainable manufacturing solution that has led to the successful production of a growing catalog of natural products and high-value chemicals. However, development at industrial levels has been hindered by the large resource investments required. Here we present an integrated Design-Build-Test-Learn (DBTL) pipeline for the discovery and optimization of biosynthetic pathways, which is designed to be compound agnostic and automated throughout. We initially applied the pipeline for the production of the flavonoid (2S)-pinocembrin in Escherichia coli, to demonstrate rapid iterative DBTL cycling with automation at every stage. In this case, application of two DBTL cycles successfully established a production pathway improved by 500-fold, with competitive titers up to 88 mg L-1. The further application of the pipeline to optimize an alkaloids pathway demonstrates how it could facilitate the rapid optimization of microbial strains for production of any chemical compound of interest.

A Toolbox for Diverse Oxyfunctionalisation of Monoterpenes.

The successful implementation of synthetic biology for chemicals biosynthesis relies on the availability of large libraries of well-characterized enzymatic building blocks. Here we present a scalable pipeline that applies the methodology of synthetic biology itself to bootstrap the creation of such a library. By designing and building a cytochrome P450 enzyme collection and testing it in a custom-made untargeted GC/MS-metabolomics-based approach, we were able to rapidly create and characterize a comprehensive enzyme library for the controlled oxyfunctionalisation of terpene scaffolds with a wide range of activities and selectivities towards several monoterpenes. This novel resource can now be used to access the extensive chemical diversity of terpenoids by pathway engineering and the assembly of biocatalytic cascades to subsequently produce libraries of oxygenated terpenoids and their derivatives for diverse applications, including drug discovery.

Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

The realization of a synthetic biology approach to microbial (1R,2S,5R)-(-)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

LipSpin: A New Bioinformatics Tool for Quantitative 1H NMR Lipid Profiling.

The structural similarity among lipid species and the low sensitivity and spectral resolution of nuclear magnetic resonance (NMR) have traditionally hampered the routine use of 1H NMR lipid profiling of complex biological samples in metabolomics, which remains mostly manual and lacks freely available bioinformatics tools. However, 1H NMR lipid profiling provides fast quantitative screening of major lipid classes (fatty acids, glycerolipids, phospholipids, and sterols) and some individual species and has been used in several clinical and nutritional studies, leading to improved risk prediction models. In this Article, we present LipSpin, a free and open-source bioinformatics tool for quantitative 1H NMR lipid profiling. LipSpin implements a constrained line shape fitting algorithm based on voigt profiles and spectral templates from spectra of lipid standards, which automates the analysis of severely overlapped spectral regions and lipid signals with complex coupling patterns. LipSpin provides the most detailed quantification of fatty acid families and choline phospholipids in serum lipid samples by 1H NMR to date. Moreover, analytical and clinical results using LipSpin quantifications conform with other techniques commonly used for lipid analysis.

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation.

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.

Null diffusion-based enrichment for metabolomics data.

Metabolomics experiments identify metabolites whose abundance varies as the conditions under study change. Pathway enrichment tools help in the identification of key metabolic processes and in building a plausible biological explanation for these variations. Although several methods are available for pathway enrichment using experimental evidence, metabolomics does not yet have a comprehensive overview in a network layout at multiple molecular levels. We propose a novel pathway enrichment procedure for analysing summary metabolomics data based on sub-network analysis in a graph representation of a reference database. Relevant entries are extracted from the database according to statistical measures over a null diffusive process that accounts for network topology and pathway crosstalk. Entries are reported as a sub-pathway network, including not only pathways, but also modules, enzymes, reactions and possibly other compound candidates for further analyses. This provides a richer biological context, suitable for generating new study hypotheses and potential enzymatic targets. Using this method, we report results from cells depleted for an uncharacterised mitochondrial gene using GC and LC-MS data and employing KEGG as a knowledge base. Partial validation is provided with NMR-based tracking of 13C glucose labelling of these cells.