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Folic acid (FA) is well known to prevent neural tube defects (NTDs), but we do not know why many human NTD cases still remain refractory to FA supplementation. Here, we investigate how the DNA demethylase TET1 interacts with maternal FA status to regulate mouse embryonic brain development. We determined that cranial NTDs display higher penetrance in non-inbred than in inbred Tet1 -/- embryos and are resistant to FA supplementation across strains. Maternal diets that are either too rich or deficient in FA are linked to an increased incidence of cranial deformities in wild type and Tet1 +/- offspring and to altered DNA hypermethylation in Tet1 -/- embryos, primarily at neurodevelopmental loci. Excess FA in Tet1 -/- embryos results in phospholipid metabolite loss and reduced expression of multiple membrane solute carriers, including a FA transporter gene that exhibits increased promoter DNA methylation and thereby mimics FA deficiency. Moreover, FA deficiency reveals that Tet1 haploinsufficiency can contribute to DNA hypermethylation and susceptibility to NTDs. Overall, our study suggests that epigenetic dysregulation may underlie NTD development despite FA supplementation., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, the details of its signal transduction cascade remain elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/β hydrolase domain-containing protein 2) as an essential mPRβ co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires mPRβ. This PLA2 activity bifurcates P4 signaling by inducing clathrin-dependent endocytosis of mPRβ, resulting in the production of lipid messengers that are G-protein coupled receptors agonists. Therefore, P4 drives meiosis by inducing an ABHD2 PLA2 activity that requires both mPRβ and ABHD2 as obligate co-receptors.

The etiology of neural tube defects (NTDs) involves complex gene-environmental interactions. Folic acid (FA) prevents NTDs, but the mechanisms remain poorly understood and at least 30% of human NTDs resist the beneficial effects of FA supplementation. Here, we identify the DNA demethylase TET1 as a nexus of folate-dependent one-carbon metabolism and genetic risk factors post-neural tube closure. We determine that cranial NTDs in Tet1 -/- embryos occur at two to three times higher penetrance in genetically heterogeneous than in homogeneous genetic backgrounds, suggesting a strong impact of genetic modifiers on phenotypic expression. Quantitative trait locus mapping identified a strong NTD risk locus in the 129S6 strain, which harbors missense and modifier variants at genes implicated in intracellular endocytic trafficking and developmental signaling. NTDs across Tet1 -/- strains are resistant to FA supplementation. However, both excess and depleted maternal FA diets modify the impact of Tet1 loss on offspring DNA methylation primarily at neurodevelopmental loci. FA deficiency reveals susceptibility to NTD and other structural brain defects due to haploinsufficiency of Tet1 . In contrast, excess FA in Tet1 -/- embryos drives promoter DNA hypermethylation and reduced expression of multiple membrane solute transporters, including a FA transporter, accompanied by loss of phospholipid metabolites. Overall, our study unravels interactions between modified maternal FA status, Tet1 gene dosage and genetic backgrounds that impact neurotransmitter functions, cellular methylation and individual susceptibilities to congenital malformations, further implicating that epigenetic dysregulation may underlie NTDs resistant to FA supplementation., Competing Interests: Competing interests: All authors declare that they have no competing interests.

cAMP signaling is a well-established regulator of melanin synthesis. Two distinct cAMP signaling pathways-the transmembrane adenylyl cyclase pathway, activated primarily by the MC1R, and the soluble adenylyl cyclase (sAC) pathway-affect melanin synthesis. The sAC pathway affects melanin synthesis by regulating melanosomal pH, and the MC1R pathway affects melanin synthesis by regulating gene expression and post-translational modifications. However, whether MC1R genotype affects melanosomal pH is poorly understood. We now report that loss of function MC1R does not affect melanosomal pH. Thus, sAC signaling appears to be the only cAMP signaling pathway that regulates melanosomal pH. We also addressed whether MC1R genotype affects sAC-dependent regulation of melanin synthesis. Although sAC loss of function in wild-type human melanocytes stimulates melanin synthesis, sAC loss of function has no effect on melanin synthesis in MC1R nonfunctional human and mouse melanocytes or skin and hair melanin in e/e mice. Interestingly, activation of transmembrane adenylyl cyclases, which increases epidermal eumelanin synthesis in e/e mice, leads to enhanced production of eumelanin in sAC-knockout mice relative to that in sAC wild-type mice. Thus, MC1R- and sAC-dependent cAMP signaling pathways define distinct mechanisms that regulate melanosomal pH and pigmentation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Monochorionic-diamniotic twin pregnancies are susceptible to unique complications arising from a single placenta shared by two fetuses. Twin-twin transfusion syndrome (TTTS) is a constellation of disturbances caused by unequal blood flow within the shared placenta giving rise to a major hemodynamic imbalance between the twins. Here, we applied TTTS as a model to uncover fetal metabolic adaptations to cardiovascular stress. We compared untargeted metabolomic analyses of amniotic fluid samples from severe TTTS cases vs. singleton controls. Amniotic fluid metabolites demonstrated alterations in fatty acid, glucose, and steroid hormone metabolism in TTTS. Among TTTS cases, unsupervised principal component analysis revealed two distinct clusters of disease defined by levels of glucose metabolites, amino acids, urea, and redox status. Our results suggest that the human fetal heart can adapt to hemodynamic stress by modulating its glucose metabolism and identify potential differences in the ability of individual fetuses to respond to cardiovascular stress., Competing Interests: Dr. Papanna reported receiving personal fees from UpToDate., (© 2023 The Authors.)

Mitochondrial diseases are a heterogeneous group of monogenic disorders that result from impaired oxidative phosphorylation (OXPHOS). As neuromuscular tissues are highly energy-dependent, mitochondrial diseases often affect skeletal muscle. Although genetic and bioenergetic causes of OXPHOS impairment in human mitochondrial myopathies are well established, there is a limited understanding of metabolic drivers of muscle degeneration. This knowledge gap contributes to the lack of effective treatments for these disorders. Here, we discovered fundamental muscle metabolic remodeling mechanisms shared by mitochondrial disease patients and a mouse model of mitochondrial myopathy. This metabolic remodeling is triggered by a starvation-like response that evokes accelerated oxidation of amino acids through a truncated Krebs cycle. While initially adaptive, this response evolves in an integrated multiorgan catabolic signaling, lipid store mobilization, and intramuscular lipid accumulation. We show that this multiorgan feed-forward metabolic response involves leptin and glucocorticoid signaling. This study elucidates systemic metabolic dyshomeostasis mechanisms that underlie human mitochondrial myopathies and identifies potential new targets for metabolic intervention., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9 D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9 WT (hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation., (© 2023. The Author(s).)

Valproic acid (VPA, valproate, Depakote) is a commonly used anti-seizure medication (ASM) in the treatment of epilepsy and a variety of other neurological disorders. While VPA and other ASMs are efficacious for management of seizures, they also increase the risk for adverse pregnancy outcomes, including neural tube defects (NTDs). Thus, the utility of these drugs during pregnancy and in women of childbearing potential presents a continuing public health challenge. Elucidating the underlying genetic or metabolic risk factors for VPA-affected pregnancies may lead to development of non-teratogenic ASMs, novel prevention strategies, or more targeted methods for managing epileptic pregnancies. To address this challenge, we performed unbiased, whole embryo metabolomic screening of E8.5 mouse embryos from two inbred strains with differential susceptibility to VPA-induced NTDs. We identified metabolites of differential abundance between the two strains, both in response to VPA exposure and in the vehicle controls. Notable enriched pathways included lipid metabolism, carnitine metabolism, and several amino acid pathways, especially cysteine and methionine metabolism. There also was increased abundance of ω-oxidation products of VPA in the more NTD-sensitive strain, suggesting differential metabolism of the drug. Finally, we found significantly reduced levels of hypotaurine in the susceptible strain regardless of VPA status. Based on this information, we hypothesized that maternal supplementation with L-carnitine (400 mg/kg), coenzyme A (200 mg/kg), or hypotaurine (350 mg/kg) would reduce VPA-induced NTDs in the sensitive strain and found that administration of hypotaurine prior to VPA exposure significantly reduced the occurrence of NTDs by close to one-third compared to controls. L-carnitine and coenzyme A reduced resorption rates but did not significantly reduce NTD risk in the sensitive strain. These results suggest that genetic variants or environmental exposures influencing embryonic hypotaurine status may be factors in determining risk for adverse pregnancy outcomes when managing the health care needs of pregnant women exposed to VPA or other ASMs., Competing Interests: RF, BW, and RC formerly held positions in TeratOmic Consulting LLC, a now defunct organization. RF also receives travel funds to attend editorial board meetings of the journal, Reproductive and Developmental Medicine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Steele, Lin, Chen, Wlodarczyk, Chen, Attarwala, Venkatesalu, Cabrera, Gross and Finnell.)

Mitochondrial dysfunction and aberrant glycolysis are hallmarks of human clear cell renal cell carcinoma (ccRCC). Whereas glycolysis is thoroughly studied, little is known about the mitochondrial contribution to the pathology of ccRCC. Mitochondrial Ndufa4l2 is predictive of poor survival of ccRCC patients, and in kidney cancer cell lines the protein supports proliferation and colony formation. Its role in ccRCC, however, remains enigmatic. We utilized our established ccRCC model, termed Transgenic Cancer of the Kidney (TRACK), to generate a novel genetically engineered mouse model in which dox-regulated expression of an shRNA decreases Ndufa4l2 levels specifically in the renal proximal tubules (PT). This targeted knockdown of Ndufa4l2 reduced the accumulation of neutral renal lipid and was associated with decreased levels of the ccRCC markers carbonic anhydrase 9 (CA9) and Enolase 1 (ENO1). These findings suggest a link between mitochondrial dysregulation (i.e. high levels of Ndufa4l2), lipid accumulation, and the expression of ccRCC markers ENO1 and CA9, and demonstrate that lipid accumulation and ccRCC development can potentially be attenuated by inhibiting Ndufa4l2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Laursen, Chen, Khani, Attarwala, Gross, Dow, Nanus and Gudas.)

Nonalcohol-associated fatty liver disease (NAFLD) is characterized by excessive hepatic accumulation of fat that can progress to steatohepatitis, and currently, therapeutic options are limited. Using a high-fat diet (HFD) mouse model of NAFLD, we determined the effects of the synthetic retinoid, AC261066, a selective retinoic acid receptor β2 (RARβ2) agonist, on the global liver transcriptomes and metabolomes of mice with dietary-induced obesity (DIO) using genome-wide RNA-seq and untargeted metabolomics. We found that AC261066 limits mRNA increases in several presumptive NAFLD driver genes, including Pklr, Fasn, Thrsp, and Chchd6. Importantly, AC261066 limits the increases in the transcript and protein levels of KHK, a key enzyme for fructose metabolism, and causes multiple changes in liver metabolites involved in fructose metabolism. In addition, in cultured murine hepatocytes, where exposure to fructose and palmitate results in a profound increase in lipid accumulation, AC261066 limits this lipid accumulation. Importantly, we demonstrate that in a human hepatocyte cell line, RARβ is required for the inhibitory effects of AC261066 on palmitate-induced lipid accumulation. Finally, our data indicate that AC261066 inhibits molecular events underpinning fibrosis and exhibits anti-inflammatory effects. In conclusion, changes in the transcriptome and metabolome indicate that AC261066 affects molecular changes underlying multiple aspects of NAFLD, including steatosis and fibrosis. Therefore, we suggest that AC261066 may have potential as an effective therapy for NAFLD., Competing Interests: Conflict of interest Weill Cornell Medicine (WCM) has filed patents on intellectual property in this manuscript and these were licensed to Sveikatal, Inc. L. J. G. and X.-H. T. are founders and have financial interests in Sveikatal, Inc. M. M., A. R., C. L., J. J., T. Z., and S. S. G. report no conflicts of interest associated with this publication. This does not alter our adherence to policies on sharing data and materials., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Noninvasive biomarkers of kidney allograft status can help minimize the need for standard of care kidney allograft biopsies. Metabolites that are measured in the urine may inform about kidney function and health status, and potentially identify rejection events. To test these hypotheses, we conducted a metabolomics study of biopsy-matched urine cell-free supernatants from kidney allograft recipients who were diagnosed with two major types of acute rejections and no-rejection controls. Non-targeted metabolomics data for 674 metabolites and 577 unidentified molecules, for 192 biopsy-matched urine samples, were analyzed. Univariate and multivariate analyses identified metabolite signatures for kidney allograft rejection. The replicability of a previously developed urine metabolite signature was examined. Our study showed that metabolite profiles can serve as biomarkers for discriminating rejection biopsies from biopsies without rejection features, but also revealed a role of estimated Glomerular Filtration Rate (eGFR) as a major confounder of the metabolite signal.

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography. Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U- 13 C] L-tyrosine using liquid chromatography-mass spectrometry. Using this method, we confirmed the previous reports of the differences in melanin synthesis between melanocytes derived from individuals with different skin colors and MC1R genotype and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-3,4-dihydroxyphenylalanine, an important metabolite of L-tyrosine, is differentially controlled by multiple factors. Because L-tyrosine fate tracing is compatible with untargeted liquid chromatography-mass spectrometry‒based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Serine is a nonessential amino acid generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. In addition, cancer cells can harness exogenous serine to enhance their metabolism and proliferation. Here we tested the relative contributions of exogenous and endogenous sources of serine on the biology of colorectal cancer. In murine tumors, Apc status was identified as a determinant of the expression of genes controlling serine synthesis. In patient samples, PSAT1 was overexpressed in both colorectal adenomas and adenocarcinomas. Combining genetic deletion of PSAT1 with exogenous serine deprivation maximally suppressed the proliferation of colorectal cancer cells and induced profound metabolic defects including diminished nucleotide production. Inhibition of serine synthesis enhanced the transcriptional changes following exogenous serine removal as well as alterations associated with DNA damage. Both loss of PSAT1 and removal of serine from the diet were necessary to suppress colorectal cancer xenograft growth and enhance the antitumor activity of 5-fluorouracil (5-FU). Restricting endogenous and exogenous serine in vitro augmented 5-FU-induced cell death, DNA damage, and metabolic perturbations, likely accounting for the observed antitumor effect. Collectively, our results suggest that both endogenous and exogenous sources of serine contribute to colorectal cancer growth and resistance to 5-FU. SIGNIFICANCE: These findings provide insights into the metabolic requirements of colorectal cancer and reveal a novel approach for its treatment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2275/F1.large.jpg., (©2021 American Association for Cancer Research.)

Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302 . Predicted miR-302 targets Pfkp , Pfkfb3 , and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.

Amyotrophic lateral sclerosis is a disease characterized by progressive paralysis and death. Most ALS-cases are sporadic (sALS) and patient heterogeneity poses challenges for effective therapies. Applying metabolite profiling on 77-sALS patient-derived-fibroblasts and 43-controls, we found ~25% of sALS cases (termed sALS-1) are characterized by transsulfuration pathway upregulation, where methionine-derived-homocysteine is channeled into cysteine for glutathione synthesis. sALS-1 fibroblasts selectively exhibited a growth defect under oxidative conditions, fully-rescued by N-acetylcysteine (NAC). [U 13 C]-glucose tracing showed transsulfuration pathway activation with accelerated glucose flux into the Krebs cycle. We established a four-metabolite support vector machine model predicting sALS-1 metabotype with 97.5% accuracy. Both sALS-1 metabotype and growth phenotype were validated in an independent cohort of sALS cases. Importantly, plasma metabolite profiling identified a system-wide cysteine metabolism perturbation as a hallmark of sALS-1. Findings reveal that sALS patients can be stratified into distinct metabotypes with differential sensitivity to metabolic stress, providing novel insights for personalized therapy., Competing Interests: Declaration of Competing Interest Nothing to report., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer's disease (AD). However, the reason for the association between APOE4 and AD remains unclear. While much of the research has focused on the ability of the apoE4 protein to increase the aggregation and decrease the clearance of Aβ, there is also an abundance of data showing that APOE4 negatively impacts many additional processes in the brain, including bioenergetics. In order to gain a more comprehensive understanding of APOE4's role in AD pathogenesis, we performed a transcriptomics analysis of APOE4 vs. APOE3 expression in the entorhinal cortex (EC) and primary visual cortex (PVC) of aged APOE mice. This study revealed EC-specific upregulation of genes related to oxidative phosphorylation (OxPhos). Follow-up analysis utilizing the Seahorse platform showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC of these mice. Additional studies, as well as the original transcriptomics data, suggest that multiple bioenergetic pathways are differentially regulated by APOE4 expression in the EC of aged APOE mice in order to increase the mitochondrial coupling efficiency in this region. Given the importance of the EC as one of the first regions to be affected by AD pathology in humans, the observation that the EC is susceptible to differential bioenergetic regulation in response to a metabolic stressor such as APOE4 may point to a causative factor in the pathogenesis of AD.

The accumulation of lipids is a hallmark of human clear cell renal cell carcinoma (ccRCC). Advanced ccRCC tumors frequently show increased lipid biosynthesis, but the regulation of lipid metabolism in early stage ccRCC tumors has not been studied. Here, we performed combined transcriptomics and metabolomics on a previously characterized transgenic mouse model (TRAnsgenic Cancer of the Kidney, TRACK) of early stage ccRCC. We found that in TRACK kidneys, HIF1α activation increases transcripts of lipid receptors (Cd36, ACVRL1), lipid storage genes (Hilpda and Fabp7), and intracellular levels of essential fatty acids, including linoleic acid and linolenic acid. Feeding the TRACK mice a high-fat diet enhances lipid accumulation in the kidneys. These results show that HIF1α increases the uptake and storage of dietary lipids in this early stage ccRCC model. By then analyzing early stage human ccRCC specimens, we found similar increases in CD36 transcripts and increases in linoleic and linolenic acid relative to normal kidney samples. CD36 mRNA levels decreased, while FASN transcript levels increased with increasing ccRCC tumor stage. These results suggest that an increase in the lipid biosynthesis pathway in advanced ccRCC tumors may compensate for a decreased capacity of these advanced ccRCCs to scavenge extracellular lipids., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

N 1 -methyladenosine (m 1 A) was proposed to be a highly prevalent modification in mRNA 5'UTRs based on mapping studies using an m 1 A-binding antibody. We developed a bioinformatic approach to discover m 1 A and other modifications in mRNA throughout the transcriptome by analyzing preexisting ultra-deep RNA-Seq data for modification-induced misincorporations. Using this approach, we detected appreciable levels of m 1 A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m 1 A-mapping approach to detect m 1 A at single-nucleotide resolution, and confirmed that the commonly used m 1 A antibody maps sites to the transcription-start site in mRNA 5'UTRs. However, further analysis revealed that these were false-positives caused by binding of the antibody to the m 7 G-cap. A different m 1 A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5'UTRs. These results demonstrate that high-stoichiometry m 1 A sites are exceedingly rare in mRNAs and that previous mappings of m 1 A to 5'UTRs were the result of antibody cross-reactivity to the 5' cap.

Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ROL) can be oxidized to all- trans -retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH's effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-γ (RARγ) knockout ESC lines generated by CRISPR/Cas9 and homologous recombination, we demonstrate that EtOH signals via RARγ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. We also report that EtOH-mediated increases in homeobox A1 ( Hoxa1 ) and cytochrome P450 family 26 subfamily A member 1 ( Cyp26a1 ) transcripts, direct RA target genes, require the expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1 family member A2 (Aldh1a2), suggesting that EtOH-mediated induction of Hoxa1 and Cyp26a1 requires ROL from the serum. As shown with CRISPR/Cas9 knockout lines, the retinol dehydrogenase gene Rdh10 and a functional RARE in the ROL transporter stimulated by retinoic acid 6 ( Stra6 ) gene are required for EtOH induction of Hoxa1 and Cyp26a1 We conclude that EtOH stimulates stem cell differentiation by increasing the influx and metabolism of ROL for downstream RARγ-dependent transcription. In stem cells, EtOH may shift cell fate decisions to alter developmental outcomes by increasing endogenous ROL/RA signaling via increased Stra6 expression and ROL oxidation., (© 2019 Serio et al.)

Despite rapid advances in sequencing technologies, accurately calling genetic variants present in an individual genome from billions of short, errorful sequence reads remains challenging. Here we show that a deep convolutional neural network can call genetic variation in aligned next-generation sequencing read data by learning statistical relationships between images of read pileups around putative variant and true genotype calls. The approach, called DeepVariant, outperforms existing state-of-the-art tools. The learned model generalizes across genome builds and mammalian species, allowing nonhuman sequencing projects to benefit from the wealth of human ground-truth data. We further show that DeepVariant can learn to call variants in a variety of sequencing technologies and experimental designs, including deep whole genomes from 10X Genomics and Ion Ampliseq exomes, highlighting the benefits of using more automated and generalizable techniques for variant calling.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a rare lysosomal storage disorder caused by a monogenetic deficiency of tripeptidyl peptidase-1 (TPP1). Despite knowledge that lipofuscin is the hallmark disease product, the relevant TPP1 substrate and its role in neuronal physiology/pathology is unknown. We hypothesized that untargeted metabolite profiling of cerebrospinal fluid (CSF) could be used as an effective tool to identify disease-associated metabolic disruptions in CLN2 disease, offering the potential to identify biomarkers that inform on disease severity and progression. Accordingly, a mass spectrometry-based untargeted metabolite profiling approach was employed to differentiate CSF from normal vs. CLN2 deficient individuals. Of 1,433 metabolite features surveyed, 29 linearly correlated with currently employed disease severity scores. With tandem mass spectrometry 8 distinct metabolite identities were structurally confirmed based on retention time and fragmentation pattern matches, vs. standards. These putative CLN2 biomarkers include 7 acetylated species - all attenuated in CLN2 compared to controls. Because acetate is the major bioenergetic fuel for support of mitochondrial respiration, deficient acetylated species in CSF suggests a brain energy defect that may drive neurodegeneration. Targeted analysis of these metabolites in CSF of CLN2 patients offers a powerful new approach for monitoring CLN2 disease progression and response to therapy.

Using molecular, biochemical, and untargeted stable isotope tracing approaches, we identify a previously unappreciated glutamine-derived α-ketoglutarate (αKG) energy-generating anaplerotic flux to be critical in mitochondrial DNA (mtDNA) mutant cells that harbor human disease-associated oxidative phosphorylation defects. Stimulating this flux with αKG supplementation enables the survival of diverse mtDNA mutant cells under otherwise lethal obligatory oxidative conditions. Strikingly, we demonstrate that when residual mitochondrial respiration in mtDNA mutant cells exceeds 45% of control levels, αKG oxidative flux prevails over reductive carboxylation. Furthermore, in a mouse model of mitochondrial myopathy, we show that increased oxidative αKG flux in muscle arises from enhanced alanine synthesis and release into blood, concomitant with accelerated amino acid catabolism from protein breakdown. Importantly, in this mouse model of mitochondriopathy, muscle amino acid imbalance is normalized by αKG supplementation. Taken together, our findings provide a rationale for αKG supplementation as a therapeutic strategy for mitochondrial myopathies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Abnormal folate one-carbon metabolism (FOCM) is implicated in neural tube defects (NTDs), severe malformations of the nervous system. MTHFR mediates unidirectional transfer of methyl groups from the folate cycle to the methionine cycle and, therefore, represents a key nexus in partitioning one-carbon units between FOCM functional outputs. Methionine cycle inhibitors prevent neural tube closure in mouse embryos. Similarly, the inability to use glycine as a one-carbon donor to the folate cycle causes NTDs in glycine decarboxylase (Gldc)-deficient embryos. However, analysis of Mthfr-null mouse embryos shows that neither S-adenosylmethionine abundance nor neural tube closure depend on one-carbon units derived from embryonic or maternal folate cycles. Mthfr deletion or methionine treatment prevents NTDs in Gldc-null embryos by retention of one-carbon units within the folate cycle. Overall, neural tube closure depends on the activity of both the methionine and folate cycles, but transfer of one-carbon units between the cycles is not necessary., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer's disease (AD). However, the reason APOE4 is associated with increased AD risk remains a source of debate. Neuronal hyperactivity is an early phenotype in both AD mouse models and in human AD, which may play a direct role in the pathogenesis of the disease. Here, we have identified an APOE4-associated hyperactivity phenotype in the brains of aged APOE mice using four complimentary techniques-fMRI, in vitro electrophysiology, in vivo electrophysiology, and metabolomics-with the most prominent hyperactivity occurring in the entorhinal cortex. Further analysis revealed that this neuronal hyperactivity is driven by decreased background inhibition caused by reduced responsiveness of excitatory neurons to GABAergic inhibitory inputs. Given the observations of neuronal hyperactivity in prodromal AD, we propose that this APOE4-driven hyperactivity may be a causative factor driving increased risk of AD among APOE4 carriers.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N 6 ,2'-O-dimethyladenosine (m 6 A m ), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m 6 A m we find that m 6 A m -initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m 6 A m -initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m 6 A m is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m 6 A m rather than N 6 -methyladenosine (m 6 A), and reduces the stability of m 6 A m mRNAs. Together, these findings show that the methylation status of m 6 A m in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer., Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39., Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = 006) and iNOS expression (P = 003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor N G -methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = 04 and P = 02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1., Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer., (© The Author 2016. Published by Oxford University Press.)

Rationale: Waterpipes, also called hookahs, are currently used by millions of people worldwide. Despite the increasing use of waterpipe smoking, there is limited data on the health effects of waterpipe smoking and there are no federal regulations regarding its use., Objectives: To assess the effects of waterpipe smoking on the human lung using clinical and biological parameters in young, light-use waterpipe smokers., Methods: We assessed young, light-use, waterpipe-only smokers in comparison with lifelong nonsmokers using clinical parameters of cough and sputum scores, lung function, and chest high-resolution computed tomography as well as biological parameters of lung epithelial lining fluid metabolome, small airway epithelial (SAE) cell differential and transcriptome, alveolar macrophage transcriptome, and plasma apoptotic endothelial cell microparticles., Measurements and Main Results: Compared with nonsmokers, waterpipe smokers had more cough and sputum as well as a lower lung diffusing capacity, abnormal epithelial lining fluid metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and alveolar macrophage transcriptomes, and elevated levels of apoptotic endothelial cell microparticles., Conclusions: Young, light-use, waterpipe-only smokers have a variety of abnormalities in multiple lung-related biological and clinical parameters, suggesting that even limited waterpipe use has broad consequences on human lung biology and health. We suggest that large epidemiological studies should be initiated to investigate the harmful effects of waterpipe smoking.

Treatment with celecoxib, a selective COX-2 inhibitor, reduces formation of premalignant adenomatous polyps in the gastrointestinal tracts of humans and mice. In addition to its chemopreventive activity, celecoxib can exhibit antimicrobial activity. Differing bacterial profiles have been found in feces from colon cancer patients compared with those of normal subjects. Moreover, preclinical studies suggest that bacteria can modulate intestinal tumorigenesis by secreting specific metabolites. In the current study, we determined whether celecoxib treatment altered the luminal microbiota and metabolome in association with reducing intestinal polyp burden in mice. Administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APC(Min/+) mice. Treatment with celecoxib also altered select luminal bacterial populations in both APC(Min/+) and wild-type mice, including decreased Lactobacillaceae and Bifidobacteriaceae as well as increased Coriobacteriaceae Metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites linked to carcinogenesis, including glucose, amino acids, nucleotides, and lipids. Ingenuity Pathway Analysis suggested that these changes in metabolites may contribute to reduced cell proliferation. To this end, we showed that celecoxib reduced cell proliferation in the base of normal appearing ileal and colonic crypts of APC(Min/+) mice. Consistent with this finding, lineage tracing indicated that celecoxib treatment reduced the rate at which Lgr5-positive stem cells gave rise to differentiated cell types in the crypts. Taken together, these results demonstrate that celecoxib alters the luminal microbiota and metabolome along with reducing epithelial cell proliferation in mice. We hypothesize that these actions contribute to its chemopreventive activity. Cancer Prev Res; 9(9); 721-31. ©2016 AACR., Competing Interests: Edward D. Karoly is an employee of Metabolon, Inc. The other authors disclosed no potential conflicts of interest., (©2016 American Association for Cancer Research.)

De novo mutations (DNMs) are important in Autism Spectrum Disorder (ASD), but so far analyses have mainly been on the ~1.5% of the genome encoding genes. Here, we performed whole genome sequencing (WGS) of 200 ASD parent-child trios and characterized germline and somatic DNMs. We confirmed that the majority of germline DNMs (75.6%) originated from the father, and these increased significantly with paternal age only (p=4.2×10 -10 ). However, when clustered DNMs (those within 20kb) were found in ASD, not only did they mostly originate from the mother (p=7.7×10 -13 ), but they could also be found adjacent to de novo copy number variations (CNVs) where the mutation rate was significantly elevated (p=2.4×10 -24 ). By comparing DNMs detected in controls, we found a significant enrichment of predicted damaging DNMs in ASD cases (p=8.0×10 -9 ; OR=1.84), of which 15.6% (p=4.3×10 -3 ) and 22.5% (p=7.0×10 -5 ) were in the non-coding or genic non-coding, respectively. The non-coding elements most enriched for DNM were untranslated regions of genes, boundaries involved in exon-skipping and DNase I hypersensitive regions. Using microarrays and a novel outlier detection test, we also found aberrant methylation profiles in 2/185 (1.1%) of ASD cases. These same individuals carried independently identified DNMs in the ASD risk- and epigenetic- genes DNMT3A and ADNP. Our data begins to characterize different genome-wide DNMs, and highlight the contribution of non-coding variants, to the etiology of ASD.

The airway epithelium is a complex pseudostratified multicellular layer lining the tracheobronchial tree, functioning as the primary defense against inhaled environmental contaminants. The major cell types of the airway epithelium include basal, intermediate columnar, ciliated, and secretory. Basal cells (BCs) are the proliferating stem/progenitor population that differentiate into the other specialized cell types of the airway epithelium during normal turnover and repair. Given that cigarette smoke delivers thousands of xenobiotics and high levels of reactive molecules to the lung epithelial surface, we hypothesized that cigarette smoke broadly perturbs BC metabolism. To test this hypothesis, primary airway BCs were isolated from healthy nonsmokers (n = 11) and healthy smokers (n = 7) and assessed by global metabolic profiling by liquid chromatography-mass spectrometry. The analysis identified 52 significant metabolites in BCs differentially expressed between smokers and nonsmokers (P < 0.05). These changes included metabolites associated with redox pathways, energy production, and inflammatory processes. Notably, BCs from smokers exhibited altered levels of the key enzyme cofactors/substrates nicotinamide adenine dinucleotide, flavin adenine dinucleotide, acetyl coenzyme A, and membrane phospholipid levels. Consistent with the high burden of oxidants in cigarette smoke, glutathione levels were diminished, whereas 3-nitrotyrosine levels were increased, suggesting that protection of airway epithelial cells against oxidative and nitrosative stress is significantly compromised in smoker BCs. It is likely that this altered metabotype is a reflection of, and likely contributes to, the disordered biology of airway BCs consequent to the stress cigarette smoking puts on the airway epithelium.

Noninvasive diagnosis and prognostication of acute cellular rejection in the kidney allograft may help realize the full benefits of kidney transplantation. To investigate whether urine metabolites predict kidney allograft status, we determined levels of 749 metabolites in 1516 urine samples from 241 kidney graft recipients enrolled in the prospective multicenter Clinical Trials in Organ Transplantation-04 study. A metabolite signature of the ratio of 3-sialyllactose to xanthosine in biopsy specimen-matched urine supernatants best discriminated acute cellular rejection biopsy specimens from specimens without rejection. For clinical application, we developed a high-throughput mass spectrometry-based assay that enabled absolute and rapid quantification of the 3-sialyllactose-to-xanthosine ratio in urine samples. A composite signature of ratios of 3-sialyllactose to xanthosine and quinolinate to X-16397 and our previously reported urinary cell mRNA signature of 18S ribosomal RNA, CD3ε mRNA, and interferon-inducible protein-10 mRNA outperformed the metabolite signatures and the mRNA signature. The area under the receiver operating characteristics curve for the composite metabolite-mRNA signature was 0.93, and the signature was diagnostic of acute cellular rejection with a specificity of 84% and a sensitivity of 90%. The composite signature, developed using solely biopsy specimen-matched urine samples, predicted future acute cellular rejection when applied to pristine samples taken days to weeks before biopsy. We conclude that metabolite profiling of urine offers a noninvasive means of diagnosing and prognosticating acute cellular rejection in the human kidney allograft, and that the combined metabolite and mRNA signature is diagnostic and prognostic of acute cellular rejection with very high accuracy., (Copyright © 2016 by the American Society of Nephrology.)

Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative, and cardiovascular diseases-importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has often involved painstaking 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications, and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitration Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology.

COPD (chronic obstructive pulmonary disease) is defined by a fixed expiratory airflow obstruction associated with disordered airways and alveolar destruction. COPD is caused by cigarette smoking and is the third greatest cause of mortality in the US. Forced expiratory volume in 1 second (FEV1) is the only validated clinical marker of COPD, but it correlates poorly with clinical features and is not sensitive enough to predict the early onset of disease. Using LC/MS global untargeted metabolite profiling of serum samples from a well-defined cohort of healthy smokers (n = 37), COPD smokers (n = 41) and non-smokers (n = 37), we sought to discover serum metabolic markers with known and/or unknown molecular identities that are associated with early-onset COPD. A total of 1,181 distinct molecular ions were detected in 95% of sera from all study subjects and 23 were found to be differentially-expressed in COPD-smokers vs. healthy-smokers. These 23 putative biomarkers were differentially-correlated with lung function parameters and used to generate a COPD prediction model possessing 87.8% sensitivity and 86.5% specificity. In an independent validation set, this model correctly predicted COPD in 8/10 individuals. These serum biomarkers included myoinositol, glycerophopshoinositol, fumarate, cysteinesulfonic acid, a modified version of fibrinogen peptide B (mFBP), and three doubly-charged peptides with undefined sequence that significantly and positively correlate with mFBP levels. Together, elevated levels of serum mFBP and additional disease-associated biomarkers point to a role for chronic inflammation, thrombosis, and oxidative stress in remodeling of the COPD airways. Serum metabolite biomarkers offer a promising and accessible window for recognition of early-stage COPD.

Hypoxia inducible factor 1 alpha (HIF1α) is a transcription factor that is frequently stabilized and active in human clear cell renal cell carcinoma (ccRCC). We have found that constitutively active HIF1α is sufficient to cause neoplastic transformation in a murine model of ccRCC termed the TRACK model. RNA sequencing (RNAseq) and untargeted metabolomics analyses of samples from TRACK kidneys demonstrate that HIF1α activates the transcription of genes that cause increased glucose uptake, glycolysis, and lactate production, as well as a decrease in the flux of pyruvate entering the tricarboxylic acid (TCA) cycle and a decrease in oxidative phosphorylation; these changes are identical to those observed in human ccRCC samples. These studies show that a constitutively active HIF1α promotes tumorigenesis in TRACK mice by mediating a metabolic switch to aerobic glycolysis, i.e., the Warburg effect, and suggest that TRACK mice are a valid model to test novel therapies targeting metabolic changes to inhibit human ccRCC.

Introduction: GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo., Methods and Results: Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2-3 of the Gch1 gene. Gch1(fl/fl) mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1(-/-) embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1(-/-) embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA., Conclusion: These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors., Methods: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models., Results: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal., Conclusions: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington's disease (HD), while htt(-/-) mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt(-/-), extended poly-Q (Htt-Q140/7), and wild-type mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt(-/-) mESCs, including (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt(-/-) early embryonic lethality., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr(385), an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress.

The serotonin transporter (SERT) is a major regulator of serotonergic neurotransmission and anxiety-related behaviors. SERT is expressed in two alternative polyadenylation forms that differ by an evolutionarily conserved element in the 3' untranslated region of its mRNA. Expression of SERT mRNA containing the distal polyadenylation element is associated with decreased anxiety-related behaviors in mice and humans, suggesting that this element has behaviorally relevant modulatory effects on SERT expression. We have identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a protein known to integrate multiple signal transduction pathways with gene expression, as a SERT distal polyadenylation element binding protein. This interaction is functionally meaningful because genetic manipulation of hnRNPK alters expression of the SERT protein. Furthermore, the trophic factor S100β induces Src-family kinase-mediated tyrosine phosphorylation of hnRNPK and increased SERT expression. These results identify a previously unknown mechanism of regulated SERT expression and provide a putative mechanism by which the SERT distal polyadenylation element modulates anxiety-related behaviors.

Motivation: Advances in high-resolution microscopy have recently made possible the analysis of gene expression at the level of individual cells. The fixed lineage of cells in the adult worm Caenorhabditis elegans makes this organism an ideal model for studying complex biological processes like development and aging. However, annotating individual cells in images of adult C.elegans typically requires expertise and significant manual effort. Automation of this task is therefore critical to enabling high-resolution studies of a large number of genes., Results: In this article, we describe an automated method for annotating a subset of 154 cells (including various muscle, intestinal and hypodermal cells) in high-resolution images of adult C.elegans. We formulate the task of labeling cells within an image as a combinatorial optimization problem, where the goal is to minimize a scoring function that compares cells in a test input image with cells from a training atlas of manually annotated worms according to various spatial and morphological characteristics. We propose an approach for solving this problem based on reduction to minimum-cost maximum-flow and apply a cross-entropy-based learning algorithm to tune the weights of our scoring function. We achieve 84% median accuracy across a set of 154 cell labels in this highly variable system. These results demonstrate the feasibility of the automatic annotation of microscopy-based images in adult C.elegans.

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an "uremic toxin" was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.

Background: We have recently documented the appearance of an anti-angiogenic peptide, endorepellin, in the urine of patients with chronic allograft dysfunction (CAD)., Methods: Here, we analyzed using enzyme-linked immunosorbent assay the excretion of anti-angiogenic peptides endostatin, pigment epithelium-derived factor (PEDF) and Kruppel-like factor-2 (KLF-2), in healthy individuals, patients with stable graft function and patients with various degrees of CAD., Results: In healthy subjects and patients with CAD-0, endostatin, PEDF and KLF-2 excretions were at the level of detection. In contrast, there were significant differences between the patients with CAD-3 and CAD-0, CAD-1 and healthy controls for endostatin and CAD-0 versus CAD-3 for PEDF, but no differences in KLF-2 excretion. Receiver operating characteristic (ROC) curve analyses demonstrated a highly discriminative profile for all three biomarkers: the combination of these parameters offered 83% sensitivity and 90% specificity in distinguishing CAD-0 from CAD-1-3. The quality of these potential biomarkers of CAD was, however, highest in discriminating CAD status in biopsy-proven cases and dropped when CAD-0 was diagnosed based on clinical criteria., Conclusions: In conclusion, these findings indicate the diagnostic potential of urinary detection of endostatin, PEDF and to lesser degree KLF-2 and suggest a mechanistic role played by anti-angiogenic substances in the developing vasculopathy and vascular rarefaction in patients with CAD.

A major challenge in systems biology is integration of molecular findings for individual enzyme activities into a cohesive high-level understanding of cellular metabolism and physiology/pathophysiology. However, meaningful prediction for how a perturbed enzyme activity will globally impact metabolism in a cell, tissue or intact organisms is precluded by multiple unknowns, including in vivo enzymatic rates, subcellular distribution and pathway interactions. To address this challenge, metabolomics offers the potential to simultaneously survey changes in thousands of structurally diverse metabolites within complex biological matrices. The present study assessed the capability of untargeted plasma metabolite profiling to discover systemic changes arising from inactivation of xanthine oxidoreductase (XOR), an enzyme that catalyzes the final steps in purine degradation. Using LC-MS coupled with a multivariate statistical data analysis platform, we confidently surveyed >3,700 plasma metabolites (50-1,000 Da) for differential expression in XOR wildtype vs. mice with inactivated XOR, arising from gene deletion or pharmacological inhibition. Results confirmed the predicted derangements in purine metabolism, but also revealed unanticipated perturbations in metabolism of pyrimidines, nicotinamides, tryptophan, phospholipids, Krebs and urea cycles, and revealed kidney dysfunction biomarkers. Histochemical studies confirmed and characterized kidney failure in xor-nullizygous mice. These findings provide new insight into XOR functions and demonstrate the power of untargeted metabolite profiling for systemic discovery of direct and indirect consequences of gene mutations and drug treatments.