Comparison of imputation and imputation-free methods for statistical analysis of mass spectrometry data with missing data.

Missing values are common in high-throughput mass spectrometry data. Two strategies are available to address missing values: (i) eliminate or impute the missing values and apply statistical methods that require complete data and (ii) use statistical methods that specifically account for missing values without imputation (imputation-free methods). This study reviews the effect of sample size and percentage of missing values on statistical inference for multiple methods under these two strategies. With increasing missingness, the ability of imputation and imputation-free methods to identify differentially and non-differentially regulated compounds in a two-group comparison study declined. Random forest and k-nearest neighbor imputation combined with a Wilcoxon test performed well in statistical testing for up to 50% missingness with little bias in estimating the effect size. Quantile regression imputation accompanied with a Wilcoxon test also had good statistical testing outcomes but substantially distorted the difference in means between groups. None of the imputation-free methods performed consistently better for statistical testing than imputation methods.

The ketone body ß-hydroxybutyrate shifts microglial metabolism and suppresses amyloid-ß oligomer-induced inflammation in human microglia.

Fatty acids are metabolized by ß-oxidation within the "mitochondrial ketogenic pathway" (MKP) to generate ß-hydroxybutyrate (BHB), a ketone body. BHB can be generated by most cells but largely by hepatocytes following exercise, fasting, or ketogenic diet consumption. BHB has been shown to modulate systemic and brain inflammation; however, its direct effects on microglia have been little studied. We investigated the impact of BHB on Aß oligomer (AßO)-stimulated human iPS-derived microglia (hiMG), a model relevant to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AßO with proinflammatory activation, which was mitigated by BHB at physiological concentrations of 0.1-2 mM. AßO stimulated glycolytic transcripts, suppressed genes in the ß-oxidation pathway, and induced over-expression of AD-relevant p46Shc, an endogenous inhibitor of thiolase, actions that are expected to suppress MKP. AßO also triggered mitochondrial Ca2+ increase, mitochondrial reactive oxygen species production, and activation of the mitochondrial permeability transition pore. BHB potently ameliorated all the above mitochondrial changes and rectified the MKP, resulting in reduced inflammasome activation and recovery of the phagocytotic function impaired by AßO. These results indicate that microglia MKP can be induced to modulate microglia immunometabolism, and that BHB can remedy "keto-deficiency" resulting from MKP suppression and shift microglia away from proinflammatory mitochondrial metabolism. These effects of BHB may contribute to the beneficial effects of ketogenic diet intervention in aged mice and in human subjects with mild AD.

Early childhood exposure to environmental phenols and parabens, phthalates, organophosphate pesticides, and trace elements in association with attention deficit hyperactivity disorder (ADHD) symptoms in the CHARGE study.

BACKGROUND: A growing body of literature investigated childhood exposure to environmental chemicals in association with attention-deficit/hyperactivity disorder (ADHD) symptoms, but limited studies considered urinary mixtures of multiple chemical classes. This study examined associations of concurrent exposure to non-persistent chemicals with ADHD symptoms in children diagnosed with autism spectrum disorder (ASD), developmental delay (DD), and typical development (TD). METHODS: A total of 549 children aged 2-5 years from the Childhood Autism Risks from Genetics and Environment (CHARGE) case-control study were administered the Aberrant Behavior Checklist (ABC). This study focused on the ADHD/noncompliance subscale and its two subdomains (hyperactivity/impulsivity, inattention). Sixty-two chemicals from four classes (phenols/parabens, phthalates, organophosphate pesticides, trace elements) were quantified in child urine samples, and 43 chemicals detected in > 70% samples were used to investigate their associations with ADHD symptoms. Negative binomial regression was used for single-chemical analysis, and weighted quantile sum regression with repeated holdout validation was applied for mixture analysis for each chemical class and all chemicals. The mixture analyses were further stratified by diagnostic group. RESULTS: A phthalate metabolite mixture was associated with higher ADHD/noncompliance scores (median count ratio [CR] = 1.10; 2.5th, 97.5th percentile: 1.00, 1.21), especially hyperactivity/impulsivity (median CR = 1.09; 2.5th, 97.5th percentile: 1.00, 1.25). The possible contributors to these mixture effects were di-2-ethylhexyl phthalate (DEHP) metabolites and mono-2-heptyl phthalate (MHPP). These associations were likely driven by children with ASD as these were observed among children with ASD, but not among TD or those with DD. Additionally, among children with ASD, a mixture of all chemicals was associated with ADHD/noncompliance and hyperactivity/impulsivity, and possible contributors were 3,4-dihydroxy benzoic acid, DEHP metabolites, MHPP, mono-n-butyl phthalate, and cadmium. CONCLUSIONS: Early childhood exposure to a phthalate mixture was associated with ADHD symptoms, particularly among children with ASD. While the diverse diagnostic profiles limited generalizability, our findings suggest a potential link between phthalate exposure and the comorbidity of ASD and ADHD.

Role of the Circadian Clock and Effect of Time-Restricted Feeding in Adenine-Induced Chronic Kidney Disease.

Most physiological functions exhibit circadian rhythmicity that is partly regulated by the molecular circadian clock. Herein, we investigated the relationship between the circadian clock and chronic kidney disease (CKD). The role of the clock gene in adenine-induced CKD and the mechanisms of interaction were investigated in mice in which Bmal1, the master regulator of the clock gene, was knocked out, and Bmal1 knockout (KO) tubule cells. We also determined whether the renoprotective effect of time-restricted feeding (TRF), a dietary strategy to enhance circadian rhythm, is clock gene-dependent. The mice with CKD showed altered expression of the core clock genes with a loss of diurnal variations in renal functions and key tubular transporter gene expression. Bmal1 KO mice developed more severe fibrosis, and transcriptome profiling followed by gene ontology analysis suggested that genes associated with the cell cycle, inflammation, and fatty acid oxidation pathways were significantly affected in the mutant mice. Tubule-specific deletion of BMAL1 in HK-2 cells by CRISPR/Cas9 led to upregulation of p21 and tumor necrosis α and exacerbated epithelial-mesenchymal transition-related gene expression upon transforming growth factor ß stimulation. Finally, TRF in the mice with CKD partially restored the disrupted oscillation of the kidney clock genes, accompanied by improved cell cycle arrest and inflammation, leading to decreased fibrosis. However, the renoprotective effect of TRF was abolished in Bmal1 KO mice, suggesting that TRF is partially dependent on the clock gene. Our data demonstrate that the molecular clock system plays an important role in CKD via cell cycle regulation and inflammation. Understanding the role of the circadian clock in kidney diseases can be a new research field for developing novel therapeutic targets.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Subcellular progression of mesenchymal transition identified by two discrete synchronous cell lines derived from the same glioblastoma.

Glioblastomas (GBM) exhibit intratumoral heterogeneity of various oncogenic evolutional processes. We have successfully isolated and established two distinct cancer cell lines with different morphological and biological characteristics that were derived from the same tissue sample of a GBM. When we compared their genomic and transcriptomic characteristics, each cell line harbored distinct mutation clusters while sharing core driver mutations. Transcriptomic analysis revealed that one cell line was undergoing a mesenchymal transition process, unlike the other cell line. Furthermore, we could identify four tumor samples containing our cell line-like clusters from the publicly available single-cell RNA-seq data, and in a set of paired longitudinal GBM samples, we could confirm three pairs where the recurrent sample was enriched in the genes specific to our cell line undergoing mesenchymal transition. The present study provides direct evidence and a valuable source for investigating the ongoing process of subcellular mesenchymal transition in GBM, which has prognostic and therapeutic implications.

Glycan biomarkers of autoimmunity and bile acid-associated alterations of the human glycome: Primary biliary cirrhosis and primary sclerosing cholangitis-specific glycans.

We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.

Brain Atrophy and White Matter Damage Linked to Peripheral Bioenergetic Deficits in the Neurodegenerative Disease FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder affecting subjects (premutation carriers) with a 55-200 CGG-trinucleotide expansion in the 5'UTR of the fragile X mental retardation 1 gene (FMR1) typically after age 50. As both the presence of white matter hyperintensities (WMHs) and atrophied gray matter on magnetic resonance imaging (MRI) are linked to age-dependent decline in cognition, here we tested whether MRI outcomes (WMH volume (WMHV) and brain volume) were correlated with mitochondrial bioenergetics from peripheral blood monocytic cells in 87 carriers with and without FXTAS. As a parameter assessing cumulative damage, WMHV was correlated to both FXTAS stages and age, and brain volume discriminated between carriers and non-carriers. Similarly, mitochondrial mass and ATP production showed an age-dependent decline across all participants, but in contrast to WMHV, only FADH2-linked ATP production was significantly reduced in carriers vs. non-carriers. In carriers, WMHV negatively correlated with ATP production sustained by glucose-glutamine and FADH2-linked substrates, whereas brain volume was positively associated with the latter and mitochondrial mass. The observed correlations between peripheral mitochondrial bioenergetics and MRI findings-and the lack of correlations with FXTAS diagnosis/stages-may stem from early brain bioenergetic deficits even before overt FXTAS symptoms and/or imaging findings.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

Heritability of apolipoprotein (a) traits in two-generational African-American and Caucasian families.

Heritability of LPA allele, apo(a) isoform sizes, and isoform-associated lipoprotein(a) [Lp(a)] levels was studied in 82 Caucasian and African-American families with two parents and two children (age: 6-74 years). We determined: 1) Lp(a) levels; 2) LPA allele sizes; 3) apo(a) isoform sizes; and 4) isoform-specific apo(a) levels (ISLs), the amount of Lp(a) carried by an individual apo(a) isoform. Trait heritability was estimated by mid-parent-offspring analysis. The ethnicity-adjusted heritability estimate for Lp(a) level was 0.95. Heritability for ISLs corresponding to the smaller LPA allele in a given allele-pair was higher than that corresponding to the larger LPA allele (0.91 vs. 0.59, P = 0.017). Although not statistically different, heritability for both apo(a) isoforms (0.90 vs. 0.70) and LPA alleles (0.98 vs. 0.82) was higher for the smaller versus larger sizes. Heritability was generally lower in African-Americans versus Caucasians with a 4-fold difference for the larger LPA allele (0.25 vs. 0.94, P = 0.001). In Caucasians, an overall higher heritability pattern was noted for the older (≥47 years) versus younger (<47 years) families. In conclusion, Lp(a) level and traits associated with the smaller LPA alleles were strongly determined by genetics, although with a varying ethnic influence. Ethnic differences in heritability of the larger LPA allele warrant further investigations.

Effects of imputation on correlation: implications for analysis of mass spectrometry data from multiple biological matrices.

With expanded access to, and decreased costs of, mass spectrometry, investigators are collecting and analyzing multiple biological matrices from the same subject such as serum, plasma, tissue and urine to enhance biomarker discoveries, understanding of disease processes and identification of therapeutic targets. Commonly, each biological matrix is analyzed separately, but multivariate methods such as MANOVAs that combine information from multiple biological matrices are potentially more powerful. However, mass spectrometric data typically contain large amounts of missing values, and imputation is often used to create complete data sets for analysis. The effects of imputation on multiple biological matrix analyses have not been studied. We investigated the effects of seven imputation methods (half minimum substitution, mean substitution, k-nearest neighbors, local least squares regression, Bayesian principal components analysis, singular value decomposition and random forest), on the within-subject correlation of compounds between biological matrices and its consequences on MANOVA results. Through analysis of three real omics data sets and simulation studies, we found the amount of missing data and imputation method to substantially change the between-matrix correlation structure. The magnitude of the correlations was generally reduced in imputed data sets, and this effect increased with the amount of missing data. Significant results from MANOVA testing also were substantially affected. In particular, the number of false positives increased with the level of missing data for all imputation methods. No one imputation method was universally the best, but the simple substitution methods (Half Minimum and Mean) consistently performed poorly.

Correction to: Metabolomics profiles of patients with Wilson disease reveal a distinct metabolic signature.

In the originally published version of this article, there was an error. The metabolomics platform used for the analysis is GC-TOF-MS, Gas Chromatography Time-of-Flight Mass Spectrometry and not Hydrophilic Interaction Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry as indicated in the original version.

Metabolomics profiles of patients with Wilson disease reveal a distinct metabolic signature.

INTRODUCTION: Wilson disease (WD) is characterized by excessive intracellular copper accumulation in liver and brain due to defective copper biliary excretion. With highly varied phenotypes and a lack of biomarkers for the different clinical manifestations, diagnosis and treatment can be difficult. OBJECTIVE: The aim of the present study was to analyze serum metabolomics profiles of patients with Wilson disease compared to healthy subjects, with the goal of identifying differentially abundant metabolites as potential biomarkers for this condition. METHODS: Hydrophilic interaction liquid chromatography-quadrupole time of flight mass spectrometry was used to evaluate the untargeted serum metabolome of 61 patients with WD (26 hepatic and 25 neurologic subtypes, 10 preclinical) compared to 15 healthy subjects. We conducted analysis of covariance with potential confounders (body mass index, age, sex) as covariates and partial least-squares analysis. RESULTS: After adjusting for clinical covariates and multiple testing, we identified 99 significantly different metabolites (FDR < 0.05) between WD and healthy subjects. Subtype comparisons also revealed significantly different metabolites compared to healthy subjects: WD hepatic subtype (67), WD neurologic subtype (57), WD hepatic-neurologic combined (77), and preclinical (36). Pathway analysis revealed these metabolites are involved in amino acid metabolism, the tricarboxylic acid cycle, choline metabolism, and oxidative stress. CONCLUSIONS: Patients with WD are characterized by a distinct metabolomics profile providing new insights into WD pathogenesis and identifying new potential diagnostic biomarkers.

Kv1.3 inhibition as a potential microglia-targeted therapy for Alzheimer's disease: preclinical proof of concept.

Microglia significantly contribute to the pathophysiology of Alzheimer's disease but an effective microglia-targeted therapeutic approach is not yet available clinically. The potassium channels Kv1.3 and Kir2.1 play important roles in regulating immune cell functions and have been implicated by in vitro studies in the 'M1-like pro-inflammatory' or 'M2-like anti-inflammatory' state of microglia, respectively. We here found that amyloid-ß oligomer-induced expression of Kv1.3 and Kir2.1 in cultured primary microglia. Likewise, ex vivo microglia acutely isolated from the Alzheimer's model 5xFAD mice co-expressed Kv1.3 and Kir2.1 as well as markers traditionally associated with M1 and M2 activation suggesting that amyloid-ß oligomer induces a microglial activation state that is more complex than previously thought. Using the orally available, brain penetrant small molecule Kv1.3 blocker PAP-1 as a tool, we showed that pro-inflammatory and neurotoxic microglial responses induced by amyloid-ß oligomer required Kv1.3 activity in vitro and in hippocampal slices. Since we further observed that Kv1.3 was highly expressed in microglia of transgenic Alzheimer's mouse models and human Alzheimer's disease brains, we hypothesized that pharmacological Kv1.3 inhibition could mitigate the pathology induced by amyloid-ß aggregates. Indeed, treating APP/PS1 transgenic mice with a 5-month oral regimen of PAP-1, starting at 9 months of age, when the animals already manifest cognitive deficits and amyloid pathology, reduced neuroinflammation, decreased cerebral amyloid load, enhanced hippocampal neuronal plasticity, and improved behavioural deficits. The observed decrease in cerebral amyloid deposition was consistent with the in vitro finding that PAP-1 enhanced amyloid-ß uptake by microglia. Collectively, these results provide proof-of-concept data to advance Kv1.3 blockers to Alzheimer's disease clinical trials.

Plasma metabolites and lipids associate with kidney function and kidney volume in hypertensive ADPKD patients early in the disease course.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by gradual cyst growth and expansion, increase in kidney volume with an ultimate decline in kidney function leading to end stage renal disease (ESRD). Given the decades long period of stable kidney function while cyst growth occurs, it is important to identify those patients who will progress to ESRD. Recent data from our and other laboratories have demonstrated that metabolic reprogramming may play a key role in cystic epithelial proliferation resulting in cyst growth in ADPKD. Height corrected total kidney volume (ht-TKV) accurately reflects cyst burden and predicts future loss of kidney function. We hypothesize that specific plasma metabolites will correlate with eGFR and ht-TKV early in ADPKD, both predictors of disease progression, potentially indicative of early physiologic derangements of renal disease severity. METHODS: To investigate the predictive role of plasma metabolites on eGFR and/or ht-TKV, we used a non-targeted GC-TOF/MS-based metabolomics approach on hypertensive ADPKD patients in the early course of their disease. Patient data was obtained from the HALT-A randomized clinical trial at baseline including estimated glomerular filtration rate (eGFR) and measured ht-TKV. To identify individual metabolites whose intensities are significantly correlated with eGFR and ht-TKV, association analyses were performed using linear regression with each metabolite signal level as the primary predictor variable and baseline eGFR and ht-TKV as the continuous outcomes of interest, while adjusting for covariates. Significance was determined by Storey's false discovery rate (FDR) q-values to correct for multiple testing. RESULTS: Twelve metabolites significantly correlated with eGFR and two triglycerides significantly correlated with baseline ht-TKV at FDR q-value < 0.05. Specific significant metabolites, including pseudo-uridine, indole-3-lactate, uric acid, isothreonic acid, and creatinine, have been previously shown to accumulate in plasma and/or urine in both diabetic and cystic renal diseases with advanced renal insufficiency. CONCLUSIONS: This study identifies metabolic derangements in early ADPKD which may be prognostic for ADPKD disease progression. CLINICAL TRIAL: HALT Progression of Polycystic Kidney Disease (HALT PKD) Study A; Clinical www.clinicaltrials.gov identifier: NCT00283686; first posted January 30, 2006, last update posted March 19, 2015.

Dysregulated Choline, Methionine, and Aromatic Amino Acid Metabolism in Patients with Wilson Disease: Exploratory Metabolomic Profiling and Implications for Hepatic and Neurologic Phenotypes.

Wilson disease (WD) is a genetic copper overload condition characterized by hepatic and neuropsychiatric symptoms with a not well-understood pathogenesis. Dysregulated methionine cycle is reported in animal models of WD, though not verified in humans. Choline is essential for lipid and methionine metabolism. Defects in neurotransmitters as acetylcholine, and biogenic amines are reported in WD; however, less is known about their circulating precursors. We aimed to study choline, methionine, aromatic amino acids, and phospholipids in serum of WD subjects. Hydrophilic interaction chromatography-quadrupole time-of-flight mass spectrometry was employed to profile serum of WD subjects categorized as hepatic, neurologic, and pre-clinical. Hepatic transcript levels of genes related to choline and methionine metabolism were verified in the Jackson Laboratory toxic milk mouse model of WD (tx-j). Compared to healthy subjects, choline, methionine, ornithine, proline, phenylalanine, tyrosine, and histidine were significantly elevated in WD, with marked alterations in phosphatidylcholines and reductions in sphingosine-1-phosphate, sphingomyelins, and acylcarnitines. In tx-j mice, choline, methionine, and phosphatidylcholine were similarly dysregulated. Elevated choline is a hallmark dysregulation in WD interconnected with alterations in methionine and phospholipid metabolism, which are relevant to hepatic steatosis. The elevated phenylalanine, tyrosine, and histidine carry implications for neurologic manifestations and are worth further investigation.

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer.

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65-21-92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (n = 44 cases and n = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development.

Multivariate two-part statistics for analysis of correlated mass spectrometry data from multiple biological specimens.

MOTIVATION: High through-put mass spectrometry (MS) is now being used to profile small molecular compounds across multiple biological sample types from the same subjects with the goal of leveraging information across biospecimens. Multivariate statistical methods that combine information from all biospecimens could be more powerful than the usual univariate analyses. However, missing values are common in MS data and imputation can impact between-biospecimen correlation and multivariate analysis results. RESULTS: We propose two multivariate two-part statistics that accommodate missing values and combine data from all biospecimens to identify differentially regulated compounds. Statistical significance is determined using a multivariate permutation null distribution. Relative to univariate tests, the multivariate procedures detected more significant compounds in three biological datasets. In a simulation study, we showed that multi-biospecimen testing procedures were more powerful than single-biospecimen methods when compounds are differentially regulated in multiple biospecimens but univariate methods can be more powerful if compounds are differentially regulated in only one biospecimen. AVAILABILITY AND IMPLEMENTATION: We provide R functions to implement and illustrate our method as supplementary information CONTACT: sltaylor@ucdavis.eduSupplementary information: Supplementary data are available at Bioinformatics online.

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria.

Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steady-state flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shc-depleted mice in vivo.