Assessment of the effects of repeated freeze thawing and extended bench top processing of plasma samples using untargeted metabolomics.

INTRODUCTION: Clinical metabolomics has utility as a screen for inborn errors of metabolism (IEM) and variant classification in patients with rare disease. It is important to understand and characterize preanalytical factors that influence assay performance during patient sample testing. OBJECTIVES: To evaluate the impact of extended thawing of human EDTA plasma samples on ice prior to extraction as well as repeated freeze-thaw cycling of samples to identify compounds that are unstable prior to metabolomic analysis. METHODS: Twenty-four (24) donor EDTA plasma samples were collected and immediately frozen at - 80 °C. Twelve samples were thawed on ice and extracted for analysis at time 0, 2, 4, and 6 h. Twelve other donor samples were repeatedly thawed and frozen up to four times and analyzed at each cycle. Compound levels at each time point/freeze-thaw cycle were compared to the control samples using matched-paired t tests to identify analytes affected by each condition. RESULTS: We identified 1026 biochemicals across all samples. Incubation of thawed EDTA plasma samples on ice for up to 6 h resulted in < 1% of biochemicals changing significantly. Freeze-thaw cycles affected a greater percentage of the metabolome; ~ 2% of biochemicals changed after 3 freeze-thaw cycles. CONCLUSIONS: Our study highlights that the number and magnitude of these changes are not as widespread as other aspects of improper sample handling. In total, < 3% of the metabolome detected on our clinical metabolomics platform should be disqualified when multiple freeze-thaw cycles or extended thawing at 4 °C are performed on a given sample.

Untargeted metabolomics as an unbiased approach to the diagnosis of inborn errors of metabolism of the non-oxidative branch of the pentose phosphate pathway.

Inborn errors of metabolism (IEM) involving the non-oxidative pentose phosphate pathway (PPP) include the two relatively rare conditions, transketolase deficiency and transaldolase deficiency, both of which can be difficult to diagnosis given their non-specific clinical presentations. Current biochemical testing approaches require an index of suspicion to consider targeted urine polyol testing. To determine whether a broad-spectrum biochemical test could accurately identify a specific metabolic pattern defining IEMs of the non-oxidative PPP, we employed the use of clinical metabolomic profiling as an unbiased novel approach to diagnosis. Subjects with molecularly confirmed IEMs of the PPP were included in this study. Targeted quantitative analysis of polyols in urine and plasma samples was accomplished with chromatography and mass spectrometry. Semi-quantitative unbiased metabolomic analysis of urine and plasma samples was achieved by assessing small molecules via liquid chromatography and high-resolution mass spectrometry. Results from untargeted and targeted analyses were then compared and analyzed for diagnostic acuity. Two siblings with transketolase (TKT) deficiency and three unrelated individuals with transaldolase (TALDO) deficiency were identified for inclusion in the study. For both IEMs, targeted polyol testing and untargeted metabolomic testing on urine and/or plasma samples identified typical perturbations of the respective disorder. Additionally, untargeted metabolomic testing revealed elevations in other PPP metabolites not typically measured with targeted polyol testing, including ribonate, ribose, and erythronate for TKT deficiency and ribonate, erythronate, and sedoheptulose 7-phosphate in TALDO deficiency. Non-PPP alternations were also noted involving tryptophan, purine, and pyrimidine metabolism for both TKT and TALDO deficient patients. Targeted polyol testing and untargeted metabolomic testing methods were both able to identify specific biochemical patterns indicative of TKT and TALDO deficiency in both plasma and urine samples. In addition, untargeted metabolomics was able to identify novel biomarkers, thereby expanding the current knowledge of both conditions and providing further insight into potential underlying pathophysiological mechanisms. Furthermore, untargeted metabolomic testing offers the advantage of having a single effective biochemical screening test for identification of rare IEMs, like TKT and TALDO deficiencies, that may otherwise go undiagnosed due to their generally non-specific clinical presentations.

Fast skeletal muscle troponin activator CK-2066260 increases fatigue resistance by reducing the energetic cost of muscle contraction.

KEY POINTS: Skeletal muscle fatigue limits performance in various physical activities, with exercise intolerance being a key symptom in a broad spectrum of diseases. We investigated whether a small molecule fast skeletal troponin activator (FSTA), CK-2066260, can mitigate muscle fatigue by reducing the cytosolic free [Ca2+ ] required to produce a given submaximal force and hence decreasing the energy requirement. Isolated intact single mouse muscle fibres and rat muscles in-situ treated with CK-2066260 showed improved muscle endurance., which was accompanied by decreased ATP demand and reduced glycogen usage. CK-2066260 treatment improved in-vivo exercise capacity in healthy rats and in a rat model of peripheral artery insufficiency. In conclusion, we show that the FSTA CK-2066260 effectively counteracts muscle fatigue in rodent skeletal muscle in vitro, in situ, and in vivo. This may translate to humans and provide a promising pharmacological treatment to patients suffering from severe muscle weakness and exercise intolerance. ABSTRACT: Skeletal muscle fatigue limits performance during physical exercise and exacerbated muscle fatigue is a prominent symptom among a broad spectrum of diseases. The present study investigated whether skeletal muscle fatigue is affected by the fast skeletal muscle troponin activator (FSTA) CK-2066260, which increases myofibrillar Ca2+ sensitivity and amplifies the submaximal force response. Because more force is produced for a given Ca2+ , we hypothesized that CK-2066260 could mitigate muscle fatigue by reducing the energetic cost of muscle activation. Isolated single mouse muscle fibres were fatigued by 100 repeated 350 ms contractions while measuring force and the cytosolic free [Ca2+ ] or [Mg2+ ] ([Mg2+ ]i ). When starting fatiguing stimulation at matching forces (i.e. lower stimulation frequency with CK-2066260): force was decreased by ∼50% with and by ∼75% without CK-2066260; [Mg2+ ]i was increased by ∼10% with and ∼32% without CK-2066260, reflecting a larger decrease in [ATP] in the latter. The glycogen content in in situ stimulated rat muscles fatigued by repeated contractions at matching forces was about two times higher with than without CK-2066260. Voluntary exercise capacity, assessed by rats performing rotarod exercise and treadmill running, was improved in the presence of CK-2066260. CK-2066260 treatment also increased skeletal muscle fatigue resistance and exercise performance in a rat model of peripheral artery insufficiency. In conclusion, we demonstrate that the FSTA CK-2066260 mitigates skeletal muscle fatigue by reducing the metabolic cost of force generation.

Untargeted metabolomic profiling reveals multiple pathway perturbations and new clinical biomarkers in urea cycle disorders.

PURPOSE: Untargeted metabolomic analysis is increasingly being used in the screening and management of individuals with inborn errors of metabolism (IEM). We aimed to test whether untargeted metabolomic analysis in plasma might be useful for monitoring the disease course and management of urea cycle disorders (UCDs). METHODS: Untargeted mass spectrometry-based metabolomic analysis was used to generate z-scores for more than 900 metabolites in plasma from 48 individuals with various UCDs. Pathway analysis was used to identify common pathways that were perturbed in each UCD. RESULTS: Our metabolomic analysis in plasma identified multiple potentially neurotoxic metabolites of arginine in arginase deficiency and, thus, may have utility in monitoring the efficacy of treatment in arginase deficiency. In addition, we were also able to detect multiple biochemical perturbations in all UCDs that likely reflect clinical management, including metabolite alterations secondary to dietary and medication management. CONCLUSION: In addition to utility in screening for IEM, our results suggest that untargeted metabolomic analysis in plasma may be beneficial for monitoring efficacy of clinical management and off-target effects of medications in UCDs and potentially other IEM.

The Mycobacterium tuberculosis regulatory network and hypoxia.

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

A metabolomic map of Zellweger spectrum disorders reveals novel disease biomarkers.

PURPOSE: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown. METHODS: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds. RESULTS: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes. CONCLUSION: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.

Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic profiling.

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

Cognitive enhancement in old dogs from dietary supplementation with a nutrient blend containing arginine, antioxidants, B vitamins and fish oil.

This study focused on the hypothesis that cognitive decline in aged dogs could be attenuated by dietary supplementation with a nutrient blend consisting of antioxidants, B vitamins, fish oil and l-arginine, referred to hereafter as the Brain Protection Blend (BPB). Baseline cognitive assessment before the start of treatment was used to establish cognitively equivalent control (10·464+2·33 kg) and treatment (12·118+3·386 kg) groups of aged dogs between 9·1 and 11·5 years of age and with body condition score of 5. After an initial wash-in period, all dogs were tested over a 6-month period on cognitive test protocols that assessed four phases of a landmark discrimination learning protocol, which assessed a spatial learning skill based on utilisation of external cues, and egocentric discrimination task, which assessed spatial learning based on internal body-centred cues. The BPB-supplemented group showed significantly better performance than the controls on the landmark 1 (P=0·0446) discrimination learning tasks, and on two egocentric discrimination reversal learning tasks (P=0·005 and P=0·01, respectively). The groups did not differ significantly (P>0·10) on the landmark zero discrimination task and the egocentric discrimination learning task. These results suggest beneficial effects are positively linked to task complexity. Many of the nutrients supplemented in the BPB diet were significantly higher in plasma, including arginine, α-tocopherol, DHA and EPA. These results indicate that long-term supplementation with the BPB can have cognition-improving effects and support the use of nutritional strategies in targeting brain ageing-associated risk factors as an intervention to delay cognitive ageing.

Probing the propensity of perchlorate anions for surface solvation by infrared photodissociation spectroscopy.

We investigate the perchlorate anion, ClO4-, microhydrated with up to eight water molecules using infrared photodissociation (IRPD) spectroscopy aided by density functional theory computations. The features observed in the IRPD spectra of D2-tagged ClO4-(H2O)n with n = 0-8 in the O-H stretching (2800-3800 cm-1) and fingerprint (800-1800 cm-1) spectral ranges are assigned to vibrational modes of the solvent, i.e., free and hydrogen-bonded O-H stretching and H2O bending modes, and of the solute, i.e., perchlorate stretching modes. The splitting of the triply degenerate antisymmetric stretching mode in the bare ClO4- (1102 cm-1) upon microhydration directly reports on the symmetry of the local solvation network, while the red-shift of the hydrogen-bonded O-H stretching bands in comparison to those of free O-H oscillators (>3700 cm-1) reflects the strength of the anion-water (3550-3650 cm-1) and water-water (3240-3540 cm-1) hydrogen bonds. The stronger water-water interaction leads to an early onset of water-water hydrogen bond formation (n = 2) and eventually asymmetric solvation of ClO4-, evidenced by the population of the second and third hydration shells at n = 5 and n = 8, respectively, before the first hydration shell is completed. The present study provides a bottom-up view of the initial stages of hydrogen-bond network formation around a high surface propensity anion, and these insights are discussed in the context of recent results obtained for the water-vapor interface of perchlorate solutions.

Analyses of SLC13A5-epilepsy patients reveal perturbations of TCA cycle.

OBJECTIVE: To interrogate the metabolic profile of five subjects from three families with rare, nonsense and missense mutations in SLC13A5 and Early Infantile Epileptic Encephalopathies (EIEE) characterized by severe, neonatal onset seizures, psychomotor retardation and global developmental delay. METHODS: Mass spectrometry of plasma, CSF and urine was used to identify consistently dysregulated analytes in our subjects. RESULTS: Distinctive elevations of citrate and dysregulation of citric acid cycle intermediates, supporting the hypothesis that loss of SLC13A5 function alters tricarboxylic acid cycle (TCA) metabolism and may disrupt metabolic compartmentation in the brain. SIGNIFICANCE: Our results indicate that analysis of plasma citrate and other TCA analytes in SLC13A5 deficient patients define a diagnostic metabolic signature that can aid in diagnosing children with this disease.

Elucidation of the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profiling assay.

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.

Elevations of C14:1 and C14:2 Plasma Acylcarnitines in Fasted Children: A Diagnostic Dilemma.

OBJECTIVES: To test whether follow-up testing for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency uncovers a diagnosis in patients with elevations of C14:1 and C14:2 plasma acylcarnitines after a controlled fasting study performed for clinically suspected hypoglycemia and to compare the acylcarnitine profiles from fasted patients without VLCAD deficiency vs patients with known VLCAD deficiency to determine whether metabolite testing distinguishes these groups. STUDY DESIGN: We performed a retrospective chart review and identified 17 patients with elevated C14:1 and C14:2 plasma acylcarnitine levels after a controlled fast and with testing for VLCAD deficiency (ACADVL sequencing or fibroblast fatty acid oxidation studies). The follow-up testing in all patients was inconsistent with a diagnosis of VLCAD deficiency. We compared the plasma acylcarnitine profiles from these fasted patients vs patients with VLCAD deficiency. RESULTS: C14:1/C12:1 was significantly lower (P < .001) in fasted patients vs patients with VLCAD deficiency. Metabolomics analysis performed in 2 fasted patients and 1 patient with VLCAD deficiency demonstrated evidence for up-regulated lipolysis and ß-oxidation in the fasted state. CONCLUSIONS: Elevations of plasma C14:1 and C14:2 acylcarnitines appear to be a physiologic result of lipolysis that occurs with fasting. Both metabolomics analysis and/or C14:1/C12:1 may distinguish C14:1 elevations from physiologic fasting-induced lipolysis vs VLCAD deficiency.

Towards better metrics and policymaking for seed system development: Insights from Asia's seed industry.

Since the 1980s, many developing countries have introduced policies to promote seed industry growth and improve the delivery of modern science to farmers, often with a long-term goal of increasing agricultural productivity in smallholder farming systems. Public, private, and civil society actors involved in shaping policy designs have, in turn, developed competing narratives around how best to build an innovative and sustainable seed system, each with varying goals, values, and levels of influence. Efforts to strike a balance between these narratives have often played out in passionate discourses surrounding seed rules and regulations. As a result, however, policymakers in many countries have expressed impatience with the slow progress on enhancing the contribution of a modern seed industry to the overarching goal of increasing agricultural productivity growth. One reason for this slow progress may be that policymakers are insufficiently cognizant of the trade-offs associated with rules and regulations required to effectively govern a modern seed industry. This suggests the need for new data and analysis to improve the understanding of how seed systems function. This paper explores these issues in the context of Asia's rapidly growing seed industry, with illustrations from seed markets for maize and several other crops, to highlight current gaps in the metrics used to analyze performance, competition, and innovation. The paper provides a finite set of indicators to inform policymaking on seed system design and monitoring, and explores how these indicators can be used to inform current policy debates in the region.

The small-molecule fast skeletal troponin activator, CK-2127107, improves exercise tolerance in a rat model of heart failure.

Heart failure-mediated skeletal myopathy, which is characterized by muscle atrophy and muscle metabolism dysfunction, often manifests as dyspnea and limb muscle fatigue. We have previously demonstrated that increasing Ca(2+) sensitivity of the sarcomere by a small-molecule fast skeletal troponin activator improves skeletal muscle force and exercise performance in healthy rats and models of neuromuscular disease. The objective of this study was to investigate the effect of a novel fast skeletal troponin activator, CK-2127107 (2-aminoalkyl-5-N-heteroarylpyrimidine), on skeletal muscle function and exercise performance in rats exhibiting heart failure-mediated skeletal myopathy. Rats underwent a left anterior descending coronary artery ligation, resulting in myocardial infarction and a progressive decline in cardiac function [left anterior descending coronary artery heart failure (LAD-HF)]. Compared with sham-operated control rats, LAD-HF rat hindlimb and diaphragm muscles exhibited significant muscle atrophy. Fatigability was increased during repeated in situ isokinetic plantar flexor muscle contractions. CK-2127107 produced a leftward shift in the force-Ca(2+) relationship of skinned, single diaphragm, and extensor digitorum longus fibers. Exercise performance, which was assessed by rotarod running, was lower in vehicle-treated LAD-HF rats than in sham controls (116 ± 22 versus 193 ± 31 seconds, respectively; mean ± S.E.M.; P = 0.04). In the LAD-HF rats, a single oral dose of CK-2127107 (10 mg/kg p.o.) increased running time compared with vehicle treatment (283 ± 47 versus 116 ± 22 seconds; P = 0.0004). In summary, CK-2127107 substantially increases exercise performance in this heart failure model, suggesting that modulation of skeletal muscle function by a fast skeletal troponin activator may be a useful therapeutic in heart failure-associated exercise intolerance.

Untargeted metabolomic analysis for the clinical screening of inborn errors of metabolism.

Global metabolic profiling currently achievable by untargeted mass spectrometry-based metabolomic platforms has great potential to advance our understanding of human disease states, including potential utility in the detection of novel and known inborn errors of metabolism (IEMs). There are few studies of the technical reproducibility, data analysis methods, and overall diagnostic capabilities when this technology is applied to clinical specimens for the diagnosis of IEMs. We explored the clinical utility of a metabolomic workflow capable of routinely generating semi-quantitative z-score values for ~900 unique compounds, including ~500 named human analytes, in a single analysis of human plasma. We tested the technical reproducibility of this platform and applied it to the retrospective diagnosis of 190 individual plasma samples, 120 of which were collected from patients with a confirmed IEM. Our results demonstrate high intra-assay precision and linear detection for the majority compounds tested. Individual metabolomic profiles provided excellent sensitivity and specificity for the detection of a wide range of metabolic disorders and identified novel biomarkers for some diseases. With this platform, it is possible to use one test to screen for dozens of IEMs that might otherwise require ordering multiple unique biochemical tests. However, this test may yield false negative results for certain disorders that would be detected by a more well-established quantitative test and in its current state should be considered a supplementary test. Our findings describe a novel approach to metabolomic analysis of clinical specimens and demonstrate the clinical utility of this technology for prospective screening of IEMs.

Metabolomic profiling reveals severe skeletal muscle group-specific perturbations of metabolism in aged FBN rats.

Mammalian skeletal muscles exhibit age-related adaptive and pathological remodeling. Several muscles in particular undergo progressive atrophy and degeneration beyond median lifespan. To better understand myocellular responses to aging, we used semi-quantitative global metabolomic profiling to characterize trends in metabolic changes between 15-month-old adult and 32-month-old aged Fischer 344 × Brown Norway (FBN) male rats. The FBN rat gastrocnemius muscle exhibits age-dependent atrophy, whereas the soleus muscle, up until 32 months, exhibits markedly fewer signs of atrophy. Both gastrocnemius and soleus muscles were analyzed, as well as plasma and urine. Compared to adult gastrocnemius, aged gastrocnemius showed evidence of reduced glycolytic metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet levels of citrate and nicotinamide adenine dinucleotide were reduced, consistent with mitochondrial abnormalities. Indicative of muscle atrophy, 3-methylhistidine and free amino acids were elevated in aged gastrocnemius. The monounsaturated fatty acids oleate, cis-vaccenate, and palmitoleate also increased in aged gastrocnemius, suggesting altered lipid metabolism. Compared to gastrocnemius, aged soleus exhibited far fewer changes in carbohydrate metabolism, but did show reductions in several glycolytic intermediates, fumarate, malate, and flavin adenine dinucleotide. Plasma biochemicals showing the largest age-related increases included glycocholate, heme, 1,5-anhydroglucitol, 1-palmitoleoyl-glycerophosphocholine, palmitoleate, and creatine. These changes suggest reduced insulin sensitivity in aged FBN rats. Altogether, these data highlight skeletal muscle group-specific perturbations of glucose and lipid metabolism consistent with mitochondrial dysfunction in aged FBN rats.

Molecular differentiation of historic phage-type 80/81 and contemporary epidemic Staphylococcus aureus.

Staphylococcus aureus is a bacterial pathogen known to cause infections in epidemic waves. One such epidemic was caused by a clone known as phage-type 80/81, a penicillin-resistant strain that rose to world prominence in the late 1950s. The molecular underpinnings of the phage-type 80/81 outbreak have remained unknown for decades, nor is it understood why related S. aureus clones became epidemic in hospitals in the early 1990s. To better understand the molecular basis of these epidemics, we sequenced the genomes of eight S. aureus clinical isolates representative of the phage-type 80/81 clone, the Southwest Pacific clone [a community-associated methicillin-resistant S. aureus (MRSA) clone], and contemporary S. aureus clones, all of which are genetically related and belong to the same clonal complex (CC30). Genome sequence analysis revealed that there was coincident divergence of these clones from a recent common ancestor, a finding that resolves controversy about the evolutionary history of the lineage. Notably, we identified nonsynonymous SNPs in genes encoding accessory gene regulator C (agrC) and α-hemolysin (hla)--molecules important for S. aureus virulence--that were present in virtually all contemporary CC30 hospital isolates tested. Compared with the phage-type 80/81 and Southwest Pacific clones, contemporary CC30 hospital isolates had reduced virulence in mouse infection models, the result of SNPs in agrC and hla. We conclude that agr and hla (along with penicillin resistance) were essential for world dominance of phage-type 80/81 S. aureus, whereas key SNPs in contemporary CC30 clones restrict these pathogens to hospital settings in which the host is typically compromised.

Integrating Genome Sequencing and Untargeted Metabolomics in Monozygotic Twins with a Rare Complex Neurological Disorder.

Multi-omics approaches, which integrate genomics, transcriptomics, proteomics, and metabolomics, have emerged as powerful tools in the diagnosis of rare diseases. We used untargeted metabolomics and whole-genome sequencing (WGS) to gain a more comprehensive understanding of a rare disease with a complex presentation affecting female twins from a consanguineous family. The sisters presented with polymicrogyria, a Dandy-Walker malformation, respiratory distress, and multiorgan dysfunctions. Through WGS, we identified two rare homozygous variants in both subjects, a pathogenic variant in ADGRG1(p.Arg565Trp) and a novel variant in CNTNAP1(p.Glu910Val). These genes have been previously associated with autosomal recessive polymicrogyria and hypomyelinating neuropathy with/without contractures, respectively. The twins exhibited symptoms that overlapped with both of these conditions. The results of the untargeted metabolomics analysis revealed significant metabolic perturbations relating to neurodevelopmental abnormalities, kidney dysfunction, and microbiome. The significant metabolites belong to essential pathways such as lipids and amino acid metabolism. The identification of variants in two genes, combined with the support of metabolic perturbation, demonstrates the rarity and complexity of this phenotype and provides valuable insights into its underlying mechanisms.

Environmental and temporal factors affecting pediatric orthopaedic injuries.

Anecdotal evidence suggests that temporal and environmental factors may contribute to the variance in the volume of pediatric orthopaedic injuries (POI), and knowing the effect of these factors could help in the allocation of resources and personnel in pediatric emergency rooms. All POI presenting to a level 1 pediatric emergency department over a 3-year period were reviewed. Environmental data, including minimal, maximal, and average daily temperatures; amount of precipitation; and lunar phase, were obtained for the study region, as were day of the week and month and season of the year. Multiple logistic regression determined which variable or combination of variables might affect the rate of POI. In the 6770 POI seen over the study period (average 6.2 per day), the day of the week and lunar phase had no effect on the rate of POI, the presence of precipitation lowered the rate slightly, and temperature had a dramatic effect, with the highest number of injuries occurring in the average daily temperature range of 70° to 79°. Winter months of November through February had the lowest rates of POI, while May had the highest.

A Case Study of Dysfunctional Nicotinamide Metabolism in a 20-Year-Old Male.

We present a case study of a 20-year-old male with an unknown neurodegenerative disease who was referred to the Undiagnosed Diseases Network Vanderbilt Medical Center site. A previous metabolic panel showed that the patient had a critical deficiency in nicotinamide intermediates that are generated during the biosynthesis of NAD(H). We followed up on these findings by evaluating the patient's ability to metabolize nicotinamide. We performed a global metabolic profiling analysis of plasma samples that were collected: (1) under normal fed conditions (baseline), (2) after the patient had fasted, and (3) after he was challenged with a 500 mg nasogastric tube bolus of nicotinamide following the fast. Our findings showed that the patient's nicotinamide N-methyltransferase (NNMT), a key enzyme in NAD(H) biosynthesis and methionine metabolism, was not functional under normal fed or fasting conditions but was restored in response to the nicotinamide challenge. Altered levels of metabolites situated downstream of NNMT and in neighboring biochemical pathways provided further evidence of a baseline defect in NNMT activity. To date, this is the only report of a critical defect in NNMT activity manifesting in adulthood and leading to neurodegenerative disease. Altogether, this study serves as an important reference in the rare disease literature and also demonstrates the utility of metabolomics as a diagnostic tool for uncharacterized metabolic diseases.