Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

Development of a Standardized Bone Marrow Procedure Training and Competency Toolkit for Advanced Practice Providers in a Large Community Oncology Practice.

Bone marrow procedures are a common diagnostic tool utilized in hematology/oncology and can be completed in the office by trained clinicians. Currently, there are limited guidelines for appropriate training and competency for bone marrow procedures performed by advanced practice providers (APPs) in a community oncology practice setting. The need to create a standardized training and competency protocol for APPs in this setting was recognized. A comprehensive, standardized educational and procedural toolkit was created. The creation of a comprehensive training toolkit for APPs in the community oncology practice setting helps to ensure a high standard of procedural proficiency and consistency among individual providers and practices. The creation of such an extensive toolkit is time consuming. By adopting and standardizing toolkits such as this one, community hematology/oncology practices can ensure the delivery of high-quality patient care by highly trained and proficient APPs.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size.

The cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where it promoted neuritogenesis in immature human neurons. Super-resolution microscopy revealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by autism spectrum disorder (ASD)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.

Brain-synthesized oestrogens regulate cortical migration in a sexually divergent manner.

Oestrogens play an important role in brain development where they have been implicated in controlling various cellular processes. Several lines of evidence have been presented showing that oestrogens can be synthesized locally within the brain. Studies have demonstrated that aromatase, the enzyme responsible for the conversion of androgens to oestrogens, is expressed during early development in both male and female cortices. Furthermore, 17ß-oestradiol has been measured in foetal brain tissue from multiple species. 17ß-oestradiol regulates neural progenitor proliferation as well as the development of early neuronal morphology. However, what role locally derived oestrogens play in regulating cortical migration and, moreover, whether these effects are the same in males and females are unknown. Here, we investigated the impact of knockdown expression of Cyp19a1, which encodes aromatase, between embryonic day (E) 14.5 and postnatal day 0 (P0) had on neural migration within the cortex. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 resulted in an increase in cells within the cortical plate, and a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. Interestingly, the opposite effect was observed in females, who displayed a significant reduction in cells migrating to the cortical plate. Together, these findings indicate that brain-derived oestrogens regulate radial migration through distinct mechanisms in males and females.

Estradiol reverses excitatory synapse loss in a cellular model of neuropsychiatric disorders.

Loss of glutamatergic synapses is thought to be a key cellular pathology associated with neuropsychiatric disorders including schizophrenia (SCZ) and major depressive disorder (MDD). Genetic and cellular studies of SCZ and MDD using in vivo and in vitro systems have supported a key role for dysfunction of excitatory synapses in the pathophysiology of these disorders. Recent clinical studies have demonstrated that the estrogen, 17ß-estradiol can ameliorate many of the symptoms experienced by patients. Yet, to date, our understanding of how 17ß-estradiol exerted these beneficial effects is limited. In this study, we have tested the hypothesis that 17ß-estradiol can restore dendritic spine number in a cellular model that recapitulates the loss of synapses associated with SCZ and MDD. Ectopic expression of wildtype, mutant or shRNA-mediated knockdown of Disrupted in Schizophrenia 1 (DISC1) reduced dendritic spine density in primary cortical neurons. Acute or chronic treatment with 17ß-estradiol increased spine density to control levels in neurons with altered DISC1 levels. In addition, 17ß-estradiol reduced the extent to which ectopic wildtype and mutant DISC1 aggregated. Furthermore, 17ß-estradiol also caused the enrichment of synaptic proteins at synapses and increased the number of dendritic spines containing PSD-95 or that overlapped with the pre-synaptic marker bassoon. Taken together, our data indicates that estrogens can restore lost excitatory synapses caused by altered DISC1 expression, potentially through the trafficking of DISC1 and its interacting partners. These data highlight the possibility that estrogens exert their beneficial effects in SCZ and MDD in part by modulating dendritic spine number.

Metabolic reprogramming and Notch activity distinguish between non-small cell lung cancer subtypes.

BACKGROUND: Previous studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking. METHODS: Publicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices. RESULTS: Analysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture. CONCLUSIONS: Our data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.

Cyto-nuclear shuttling of afadin is required for rapid estradiol-mediated modifications of histone H3.

Estrogens have been shown to rapidly regulate local signalling at synapses and within the nucleus. The result of these signalling events is to rapidly modulate synapse structure and function, as well as epigenetic mechanisms including histone modifications. Ultimately these mechanisms are thought to contribute to long-lasting changes in neural circuitry, and thus influence cognitive functions such as learning and memory. However, the mechanisms by which estrogen-mediated local synaptic and nuclear signalling events are coordinated are not well understood. In this study we have found that the scaffold protein afadin, (also known as AF-6), undergoes a bi-directional trafficking to both synaptic and nuclear compartment in response to acute 17ß-estradiol (estradiol) treatment, in mixed sex neuronal cultures derived from fetal cortex. Interestingly, nuclear accumulation of afadin was coincidental with an increase in the phosphorylation of histone H3 at serine 10 (H3S10p). This epigenetic modification is associated with the remodeling of chromatin into an open euchromatin state, allowing for transcriptional activation and related learning and memory processes. Critically, the cyto-nuclear trafficking of afadin was required for estradiol-dependent H3S10p. We further determined that nuclear accumulation of afadin is sufficient to induce phosphorylation of the mitogentic kinases ERK1/2 (pERK1/2) within the nucleus. Moreover, nuclear pERK1/2 was required for estradiol-dependent H3S10p. Taken together, we propose a model whereby estradiol induces the bi-directional trafficking of afadin to synaptic and nuclear sub-compartments. Within the nucleus, afadin is required for increased pERK1/2 which in turn is required for H3S10p. Therefore this represents a mechanism through which estrogens may be able to coordinate both synaptic and nucleosomal events within the same neuronal population.

Estradiol and the Development of the Cerebral Cortex: An Unexpected Role?

The cerebral cortex undergoes rapid folding in an "inside-outside" manner during embryonic development resulting in the establishment of six discrete cortical layers. This unique cytoarchitecture occurs via the coordinated processes of neurogenesis and cell migration. In addition, these processes are fine-tuned by a number of extracellular cues, which exert their effects by regulating intracellular signaling pathways. Interestingly, multiple brain regions have been shown to develop in a sexually dimorphic manner. In many cases, estrogens have been demonstrated to play an integral role in mediating these sexual dimorphisms in both males and females. Indeed, 17ß-estradiol, the main biologically active estrogen, plays a critical organizational role during early brain development and has been shown to be pivotal in the sexually dimorphic development and regulation of the neural circuitry underlying sex-typical and socio-aggressive behaviors in males and females. However, whether and how estrogens, and 17ß-estradiol in particular, regulate the development of the cerebral cortex is less well understood. In this review, we outline the evidence that estrogens are not only present but are engaged and regulate molecular machinery required for the fine-tuning of processes central to the cortex. We discuss how estrogens are thought to regulate the function of key molecular players and signaling pathways involved in corticogenesis, and where possible, highlight if these processes are sexually dimorphic. Collectively, we hope this review highlights the need to consider how estrogens may influence the development of brain regions directly involved in the sex-typical and socio-aggressive behaviors as well as development of sexually dimorphic regions such as the cerebral cortex.

Amyloid ß synaptotoxicity is Wnt-PCP dependent and blocked by fasudil.

INTRODUCTION: Synapse loss is the structural correlate of the cognitive decline indicative of dementia. In the brains of Alzheimer's disease sufferers, amyloid ß (Aß) peptides aggregate to form senile plaques but as soluble peptides are toxic to synapses. We previously demonstrated that Aß induces Dickkopf-1 (Dkk1), which in turn activates the Wnt-planar cell polarity (Wnt-PCP) pathway to drive tau pathology and neuronal death. METHODS: We compared the effects of Aß and of Dkk1 on synapse morphology and memory impairment while inhibiting or silencing key elements of the Wnt-PCP pathway. RESULTS: We demonstrate that Aß synaptotoxicity is also Dkk1 and Wnt-PCP dependent, mediated by the arm of Wnt-PCP regulating actin cytoskeletal dynamics via Daam1, RhoA and ROCK, and can be blocked by the drug fasudil. DISCUSSION: Our data add to the importance of aberrant Wnt signaling in Alzheimer's disease neuropathology and indicate that fasudil could be repurposed as a treatment for the disease.

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase-tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I-positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

Psychosis Risk Candidate ZNF804A Localizes to Synapses and Regulates Neurite Formation and Dendritic Spine Structure.

BACKGROUND: Variation in the gene encoding zinc finger binding protein 804A (ZNF804A) is associated with schizophrenia and bipolar disorder. Evidence suggests that ZNF804A is a regulator of gene transcription and is present in nuclear and extranuclear compartments. However, a detailed examination of ZNF804A distribution and its neuronal functions has yet to be performed. METHODS: The localization of ZNF804A protein was examined in neurons derived from human neural progenitor cells, human induced pluripotent stem cells, or in primary rat cortical neurons. In addition, small interfering RNA-mediated knockdown of ZNF804A was conducted to determine its role in neurite formation, maintenance of dendritic spine morphology, and responses to activity-dependent stimulations. RESULTS: Endogenous ZNF804A protein localized to somatodendritic compartments and colocalized with the putative synaptic markers in young neurons derived from human neural progenitor cells and human induced pluripotent stem cells. In mature rat neurons, Zfp804A, the homolog of ZNF804A, was present in a subset of dendritic spines and colocalized with synaptic proteins in specific nanodomains, as determined by super-resolution microscopy. Interestingly, knockdown of ZNF804A attenuated neurite outgrowth in young neurons, an effect potentially mediated by reduced neuroligin-4 expression. Furthermore, knockdown of ZNF804A in mature neurons resulted in the loss of dendritic spine density and impaired responses to activity-dependent stimulation. CONCLUSIONS: These data reveal a novel subcellular distribution for ZNF804A within somatodendritic compartments and a nanoscopic organization at excitatory synapses. Moreover, our results suggest that ZNF804A plays an active role in neurite formation, maintenance of dendritic spines, and activity-dependent structural plasticity.

MYC/PGC-1α Balance Determines the Metabolic Phenotype and Plasticity of Pancreatic Cancer Stem Cells.

The anti-diabetic drug metformin targets pancreatic cancer stem cells (CSCs), but not their differentiated progenies (non-CSCs), which may be related to distinct metabolic phenotypes. Here we conclusively demonstrate that while non-CSCs were highly glycolytic, CSCs were dependent on oxidative metabolism (OXPHOS) with very limited metabolic plasticity. Thus, mitochondrial inhibition, e.g., by metformin, translated into energy crisis and apoptosis. However, resistant CSC clones eventually emerged during treatment with metformin due to their intermediate glycolytic/respiratory phenotype. Mechanistically, suppression of MYC and subsequent increase of PGC-1α were identified as key determinants for the OXPHOS dependency of CSCs, which was abolished in resistant CSC clones. Intriguingly, no resistance was observed for the mitochondrial ROS inducer menadione and resistance could also be prevented/reversed for metformin by genetic/pharmacological inhibition of MYC. Thus, the specific metabolic features of pancreatic CSCs are amendable to therapeutic intervention and could provide the basis for developing more effective therapies to combat this lethal cancer.

Rapid modulation of synaptogenesis and spinogenesis by 17ß-estradiol in primary cortical neurons.

In the mammalian forebrain, the majority of excitatory synapses occur on dendritic spines. Changes in the number of these structures is important for brain development, plasticity and the refinement of neuronal circuits. The formation of excitatory synapses involves the coordinated formation of dendritic spines and targeting of multi-protein complexes to nascent connections. Recent studies have demonstrated that the estrogen 17ß-estradiol (E2) can rapidly increase the number of dendritic spines, an effect consistent with the ability of E2 to rapidly influence cognitive function. However, the molecular composition of E2-induced spines and whether these protrusions form synaptic connections has not been fully elucidated. Moreover, which estrogen receptor(s) (ER) mediate these spine-morphogenic responses are not clear. Here, we report that acute E2 treatment results in the recruitment of postsynaptic density protein 95 (PSD-95) to novel dendritic spines. In addition neuroligin 1 (Nlg-1) and the NMDA receptor subunit GluN1 are recruited to nascent synapses in cortical neurons. The presence of these synaptic proteins at nascent synapses suggests that the machinery to allow pre- and post-synapses to form connections are present in E2-induced spines. We further demonstrate that E2 treatment results in the rapid and transient activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and the mammalian target of rapamycin (mTOR) signaling pathways. However, only ERK1/2 and Akt are required for E2-mediated spinogenesis. Using synthetic receptor modulators, we further demonstrate that activation of the estrogen receptor beta (ERß) but not alpha (ERα) mimics rapid E2-induced spinogenesis and synaptogenesis. Taken together these findings suggest that in primary cortical neurons, E2 signaling via ERß, but not through ERα, is capable of remodeling neuronal circuits by increasing the number of excitatory synapses.

Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation.

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Molecular signature of rapid estrogen regulation of synaptic connectivity and cognition.

There is now a growing appreciation that estrogens are capable of rapidly activating a number of signaling cascades within the central nervous system. In addition, there are an increasing number of studies reporting that 17ß-estradiol, the major biologically active estrogen, can modulate cognition within a rapid time frame. Here we review recent studies that have begun to uncover the molecular and cellular framework which contributes to estrogens ability to rapidly modulate cognition. We first describe the mechanisms by which estrogen receptors (ERs) can couple to intracellular signaling cascades, either directly, or via the transactivation of other receptors. Subsequently, we review the evidence that estrogen can rapidly modulate both neuronal function and structure in the hippocampus and the cortex. Finally, we will discuss how estrogens may influence cognitive function through the modulation of neuronal structure, and the implications this may have on the treatment of a range of brain disorders.

Transcriptional control of GABAergic neuronal subtype identity in the thalamus.

BACKGROUND: The thalamus is often defined as the 'gateway to consciousness', a feature that is supported by the specific connectivity and electrophysiological properties of its neurons. Inhibitory GABAergic neurons are required for the dynamic gating of information passing through the thalamus. The high degree of heterogeneity among thalamic GABA neurons suggests that, during embryonic development, alternative differentiation programmes exist to guide the acquisition of inhibitory neuron subtype identity. RESULTS: Taking advantage of the accessibility of the developing chick embryo, we have used in ovo manipulations of gene expression to test the role of candidate transcription factors in controlling GABAergic neuronal subtype identity in the developing thalamus. CONCLUSIONS: In this study, we describe two alternative differentiation programmes for GABAergic neurogenesis in the thalamus and identify Helt and Dlx2 as key transcription factors that are sufficient to direct neuronal progenitors along a specific differentiation pathway at the expense of alternative lineage choices. Furthermore, we identify Calb2, a gene encoding for the GABA subtype marker calretinin as a target of the transcription factor Sox14. This work is a step forward in our understanding of how GABA neuron diversity in the thalamus is achieved during development and will help future investigation of the molecular mechanisms that lead up to the acquisition of different synaptic targets and electrophysiological features of mature thalamic inhibitory neurons.

Coordinated nuclear and synaptic shuttling of afadin promotes spine plasticity and histone modifications.

The ability of a neuron to transduce extracellular signals into long lasting changes in neuronal morphology is central to its normal function. Increasing evidence shows that coordinated regulation of synaptic and nuclear signaling in response to NMDA receptor activation is crucial for long term memory, synaptic tagging, and epigenetic signaling. Although mechanisms have been proposed for synapse-to-nuclear communication, it is unclear how signaling is coordinated at both subcompartments. Here, we show that activation of NMDA receptors induces the bi-directional and concomitant shuttling of the scaffold protein afadin from the cytosol to the nucleus and synapses. Activity-dependent afadin nuclear translocation peaked 2 h post-stimulation, was independent of protein synthesis, and occurred concurrently with dendritic spine remodeling. Moreover, activity-dependent afadin nuclear translocation coincides with phosphorylation of histone H3 at serine 10 (H3S10p), a marker of epigenetic modification. Critically, blocking afadin nuclear accumulation attenuated activity-dependent dendritic spine remodeling and H3 phosphorylation. Collectively, these data support a novel model of neuronal nuclear signaling whereby dual-residency proteins undergo activity-dependent bi-directional shuttling from the cytosol to synapses and the nucleus, coordinately regulating dendritic spine remodeling and histone modifications.

Subcortical visual shell nuclei targeted by ipRGCs develop from a Sox14+-GABAergic progenitor and require Sox14 to regulate daily activity rhythms.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) and their nuclear targets in the subcortical visual shell (SVS) are components of the non-image-forming visual system, which regulates important physiological processes, including photoentrainment of the circadian rhythm. While ipRGCs have been the subject of much recent research, less is known about their central targets and how they develop to support specific behavioral functions. We describe Sox14 as a marker to follow the ontogeny of the SVS and find that the complex forms from two narrow stripes of Dlx2-negative GABAergic progenitors in the early diencephalon through sequential waves of tangential migration. We characterize the requirement for Sox14 to orchestrate the correct distribution of neurons among the different nuclei of the network and describe how Sox14 expression is required both to ensure robustness in circadian entrainment and for masking of motor activity.

Stable isotope-resolved metabolomics and applications for drug development.

Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), during the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly cancer. The metabolome is the functional readout of the genome, functional genome, and proteome; it is also an integral partner in molecular regulations for homeostasis. The interrogation of the metabolome, or metabolomics, is now being applied to numerous diseases, largely by metabolite profiling for biomarker discovery, but also in pharmacology and therapeutics. Recent advances in stable isotope tracer-based metabolomic approaches enable unambiguous tracking of individual atoms through compartmentalized metabolic networks directly in human subjects, which promises to decipher the complexity of the human metabolome at an unprecedented pace. This knowledge will revolutionize our understanding of complex human diseases, clinical diagnostics, as well as individualized therapeutics and drug response. In this review, we focus on the use of stable isotope tracers with metabolomics technologies for understanding metabolic network dynamics in both model systems and in clinical applications. Atom-resolved isotope tracing via the two major analytical platforms, NMR and MS, has the power to determine novel metabolic reprogramming in diseases, discover new drug targets, and facilitates ADME studies. We also illustrate new metabolic tracer-based imaging technologies, which enable direct visualization of metabolic processes in vivo. We further outline current practices and future requirements for biochemoinformatics development, which is an integral part of translating stable isotope-resolved metabolomics into clinical reality.