5hmC enhances PARP trapping and restores PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells.

Mutations in BRCA1 and BRCA2 genes are the leading cause of hereditary breast and ovarian cancer. BRCA1/2-mutant cells are defective in repairing damaged DNA by homologous recombination and are characterized by hypersensitivity to PARP inhibitors. PARP inhibitors can trap PARP proteins on the chromatin, a mechanism that can contribute to the death of BRCA1/2-deficient cells.

Sphingolipid biosynthesis is essential for metabolic rewiring during T17 cell differentiation.

T helper 17 (T17) cells are implicated in autoimmune diseases, and several metabolic processes are shown to be important for their development and function. In this study, we report an essential role for sphingolipids synthesized through the de novo pathway in T17 cell development. Deficiency of SPTLC1, a major subunit of serine palmitoyl transferase enzyme complex that catalyzes the first and rate-limiting step of de novo sphingolipid synthesis, impaired glycolysis in differentiating T17 cells by increasing intracellular reactive oxygen species (ROS) through enhancement of nicotinamide adenine dinucleotide phosphate oxidase 2 activity.

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition.

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery.

Protonation-Dependent Sequencing of 5-Formylcytidine in RNA.

Chemical modification of cytidine in noncoding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remain unknown due to a lack of sensitive site-specific sequencing technologies. Here, we report a protonation-dependent sequencing reaction for the detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA.

Remodelin Is a Cryptic Assay Interference Chemotype That Does Not Inhibit NAT10-Dependent Cytidine Acetylation.

Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in ALARM NMR and proteome-wide affinity profiling assays.

SSSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis.

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown.

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability.

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.

Assessing Endogenous and Exogenous Hormone Exposures and Breast Development in a Migrant Study of Bangladeshi and British Girls.

Timing of breast development (or thelarche) and its endogenous and exogenous determinants may underlie global variation in breast cancer incidence. The study objectives were to characterize endogenous estrogen levels and bisphenol A (BPA) exposure using a migrant study of adolescent girls and test whether concentrations explained differences in thelarche by birthplace and growth environment. Estrogen metabolites (EM) and BPA-glucuronide (BPA-G) were quantified in urine spot samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) from a cross-sectional study of Bangladeshi, first- and second-generation Bangladeshi migrants to the UK, and white British girls aged 5-16 years ( = 348).

Sptlc1 is essential for myeloid differentiation and hematopoietic homeostasis.

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised.

Glutamine synthetase is necessary for sarcoma adaptation to glutamine deprivation and tumor growth.

Despite a growing body of knowledge about the genomic landscape and molecular pathogenesis of sarcomas, translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Renewed interest in altered metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a novel therapeutic strategy. In this study, we have characterized the dependency of human pediatric sarcoma cells on key metabolic substrates and identified a mechanism of adaptation to metabolic stress by examining proliferation and bioenergetic properties of rhabdomyosarcoma and Ewing sarcoma cells under varying concentrations of glucose and glutamine.

Acetylation of Cytidine in mRNA Promotes Translation Efficiency.

Generation of the "epitranscriptome" through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here, we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences.

Profiling Cytidine Acetylation with Specific Affinity and Reactivity.

The human acetyltransferase NAT10 has recently been shown to catalyze formation of N4-acetylcytidine (ac4C), a minor nucleobase known to alter RNA structure and function. In order to better understand the role of RNA acetyltransferases in biology and disease, here we report the development and application of chemical methods to study ac4C. First, we demonstrate that ac4C can be conjugated to carrier proteins using optimized protocols.

Serial Free Bisphenol A and Bisphenol A Glucuronide Concentrations in Neonates.

Objective: To determine the balance of metabolism of free bisphenol A (BPA) to the inactive conjugate, BPA glucuronide (BPAG), in neonates.

Study Design: Free BPA and BPAG concentrations were measured in 78 urine samples collected between December 2012 and August 2013 from a cohort of 44 healthy full term (≥ 37 weeks' gestation) neonates at 2 intervals (3-6 days and 7-27 days of age). A questionnaire was administered at the time of sample collection.

Urinary bisphenol A-glucuronide and postmenopausal breast cancer in Poland.

Purpose: Concerns regarding a possible link between bisphenol A (BPA) and breast cancer have been mounting, but studies in human populations are lacking. We evaluated the association between the major urinary BPA metabolite [BPA-glucuronide (BPA-G)] and postmenopausal breast cancer risk in a large population-based case-control study conducted in two cities in Poland (2000-2003); we further explored the association of BPA-G levels with known postmenopausal breast cancer risk factors in our control population.

Methods: We analyzed creatinine-adjusted urinary BPA-G levels among 575 postmenopausal cases matched on age and study site to 575 controls without breast cancer using a recently developed assay.

Prospective evaluation of serum sarcosine and risk of prostate cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

Metabolomic profiling has identified, sarcosine, a derivative of the amino acid glycine, as an important metabolite involved in the etiology or natural history of prostate cancer. We examined the association between serum sarcosine levels and risk of prostate cancer in 1122 cases (813 non-aggressive and 309 aggressive) and 1112 controls in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Sarcosine was quantified using high-throughput liquid chromatography-mass spectrometry.

Urinary free bisphenol A and bisphenol A-glucuronide concentrations in newborns.

Infants are exposed to the endocrine disruptor bisphenol A (BPA) through breast milk and baby formula. Detoxication by conjugation of BPA may be limited in infants. We demonstrate BPA exposure in 11 neonates and 1 young infant, but find no evidence of a low capacity for BPA conjugation.

Global proteomics and metabolomics in cancer biomarker discovery.

Chromatography and electrophoresis have been used for the last half-century to separate small and large molecules. Advances in MS instrumentation and techniques for sample introduction into the mass analyzer (i.e.

A reproducible and high-throughput HPLC/MS method to separate sarcosine from α- and β-alanine and to quantify sarcosine in human serum and urine.

While sarcosine was recently identified as a potential urine biomarker for prostate cancer, further studies have cast doubt on its utility to diagnose this condition. The inconsistent results may be due to the fact that alanine and sarcosine coelute on an HPLC reversed-phase column and the mass spectrometer cannot differentiate between the two isomers, since the same parent/product ions are generally used to measure them. In this study, we developed a high-throughput liquid chromatography-mass spectrometry (LC-MS) method that resolves sarcosine from alanine isomers, allowing its accurate quantification in human serum and urine.

Quantitation of free and total bisphenol A in human urine using liquid chromatography-tandem mass spectrometry.

Bisphenol A (BPA) is employed in the synthesis of polycarbonate plastics and epoxy resins and is widely used in consumer products including as a coating for the inside of almost all food and beverage containers and thermal-imaging paper. Bisphenol A is considered to have important health implications because it possesses weak estrogenic activity and can leach from storage containers resulting in its consumption by both humans and animals. It is metabolized in the body and excreted into urine as a glucuronide derivative.

Metformin prevents tobacco carcinogen--induced lung tumorigenesis.

Activation of the mammalian target of rapamycin (mTOR) pathway is an important and early event in tobacco carcinogen-induced lung tumorigenesis, and therapies that target mTOR could be effective in the prevention or treatment of lung cancer. The biguanide metformin, which is widely prescribed for the treatment of type II diabetes, might be a good candidate for lung cancer chemoprevention because it activates AMP-activated protein kinase (AMPK), which can inhibit the mTOR pathway. To test this, A/J mice were treated with oral metformin after exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Tryptophan/kynurenine metabolism in human leukocytes is independent of superoxide and is fully maintained in chronic granulomatous disease.

In chronic granulomatous disease (CGD), defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity causes reduced superoxide anion (O(2)(·)) radical production leading to frequent infections as well as granulomas and impaired wound healing indicative of excessive inflammation. Based on recent mouse studies, the lack of O(2)(·)-dependent interferon γ (IFNγ)-induced synthesis of kynurenine (kyn), an anti-inflammatory tryptophan metabolite produced by indolamine 2,3 deoxygenase (IDO), was proposed as a cause of hyperinflammation in CGD and this pathway has been considered for clinical intervention. Here, we show that IFNγ induces normal levels of kynurenine in cultures of O(2)(·)-deficient monocytes, dendritic cells, and polymorphonuclear leukocytes from gp91(PHOX)- or p47(PHOX)-deficient human CGD donors.

Rapamycin protects mice from staphylococcal enterotoxin B-induced toxic shock and blocks cytokine release in vitro and in vivo.

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-2, gamma interferon (IFN-gamma), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and T-cell proliferation.

CD4 T cell survival after intermittent interleukin-2 therapy is predictive of an increase in the CD4 T cell count of HIV-infected patients.

Administration of interleukin (IL)-2 to human immunodeficiency virus (HIV)-infected patients leads to significant increases in CD4 T cell counts. We previously have shown that IL-2 induces increased proliferation and survival of CD4 T cells. Deuterium labeling studies were undertaken to study the relationship between IL-2-induced increases in the CD4 T cell count and the effects of IL-2 on cell proliferation and survival.