Research advances from the National Institutes of Health (NIH) Intramural Research Program (IRP) are often published in high-impact journals. Read some of our recent articles:
Author: Chan K, Roberts SA, Klimczak LJ, Sterling JF, Saini N, Malc EP, Kim J, Kwiatkowski DJ, Fargo DC, Mieczkowski PA, Getz G, Gordenin DA
Journal: Nat Genet. 2015 Aug 10. doi: 10.1038/ng.3378.
Elucidation of mutagenic processes shaping cancer genomes is a fundamental problem whose solution promises insights into new treatment, diagnostic and prevention strategies. Single-strand DNA-specific APOBEC cytidine deaminase(s) are major source(s) of mutation in several cancer types. Previous indirect evidence implicated APOBEC3B as the more likely major mutator deaminase, whereas the role of APOBEC3A is not established. Using yeast models enabling the controlled generation of long single-strand genomic DNA substrates, we show that the mutation signatures of APOBEC3A and APOBEC3B are statistically distinguishable. We then apply three complementary approaches to identify cancer samples with mutation signatures resembling either APOBEC. Strikingly, APOBEC3A-like samples have over tenfold more APOBEC-signature mutations than APOBEC3B-like samples. We propose that APOBEC3A-mediated mutagenesis is much more frequent because APOBEC3A itself is highly proficient at generating DNA breaks, whose repair can trigger the formation of single-strand hypermutation substrates.
Authors: Moon AF, Gosavi RA, Kunkel TA, Pedersen LC, Bebenek K
Journal: Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):E4530-6. doi: 10.1073/pnas.1505798112.
Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol µ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol µ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol µ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol µ "skips" the first available template nucleotide, instead using the template base at the 5' end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3'-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.
Authors: Thorne PS, Mendy A, Metwali N, Salo P, Co C, Jaramillo R, Rose KM, Zeldin DC
Journal: Am J Respir Crit Care Med. 2015 Aug 10.
RATIONALE: Inhaled endotoxin induces airway inflammation and is an established risk factor for asthma. The 2005-2006 National Health and Nutrition Examination Survey (NHANES) included measures of endotoxin and allergens in homes and specific IgE to inhalant allergens.
OBJECTIVE: To understand the relationship between endotoxin exposure, asthma outcomes and sensitization status for 15 aeroallergens in a nationally representative sample.
METHODS: Participants were administered questionnaires in their homes. Reservoir dust was vacuum-sampled to generate composite bedding and bedroom floor samples. We analyzed 7450 NHANES dust and quality assurance samples for their endotoxin content using extreme quality assurance measures. Data for 6963 subjects were available making this the largest study of endotoxin exposure to date. Log-transformed endotoxin concentrations were analyzed using logistic models and forward stepwise linear regression. Analyses were weighted to provide national prevalence estimates and unbiased variances.
MAIN RESULTS: Endotoxin exposure was significantly associated with wheeze in the past 12 months, wheeze during exercise, doctor/emergency room visits for wheeze, and use of prescription medications for wheeze. Models adjusted for age, gender, race/ethnicity and poverty income ratio and stratified by allergy status showed that these relationships were not dependent upon sensitization status but were worsened among those living in poverty. Significant predictors of higher endotoxin exposures were lower family income; Hispanic ethnicity; participant age; dog(s), cat(s), cockroaches or smoker(s) in the home; and carpeted floor.
CONCLUSION: In this US nationwide representative sample, higher endotoxin exposure was significantly associated with measures of wheeze with no observed protective effect regardless of sensitization status.
Authors: Graffe M, Zenisek D, Taraska JW
Journal: J Gen Physiol. 2015 Jul;146(1):109-17. doi: 10.1085/jgp.201511396
A set of bipolar cells in the retina of goldfish contains giant synaptic terminals that can be over 10 µm in diameter. Hundreds of thousands of synaptic vesicles fill these terminals and engage in continuous rounds of exocytosis. How the cytoskeleton and other organelles in these neurons are organized to control synaptic activity is unknown. Here, we used 3-D fluorescence and 3-D electron microscopy to visualize the complex subcellular architecture of these terminals. We discovered a thick band of microtubules that emerged from the axon to loop around the terminal periphery throughout the presynaptic space. This previously unknown microtubule structure associated with a substantial population of mitochondria in the synaptic terminal. Drugs that inhibit microtubule-based kinesin motors led to accumulation of mitochondria in the axon. We conclude that this prominent microtubule band is crucial to the transport and localization of mitochondria into the presynaptic space to provide the sustained energy necessary for continuous transmitter release in these giant synaptic terminals.
Authors: Bonaventura J, Navarro G, Casadó-Anguera V, Azdad K, Rea W, Moreno E, Brugarolas M, Mallol J, Canela EI, Lluís C, Cortés A, Volkow ND, Schiffmann SN, Ferré S, Casadó V
Journal: Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3609-18. doi: 10.1073/pnas.1507704112
Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.
Authors: Schlögl M, Piaggi P, Pannacciuli N, Bonfiglio SM, Krakoff J, Thearle MS
Journal: Diabetes. 2015 Jul 16. pii: db150382. [Epub ahead of print]
Because it is unknown if 24-h energy expenditure (EE) responses to dietary extremes will identify phenotypes associated with weight regulation, the aim of this study was to determine whether such responses to fasting or overfeeding associate with future weight change. The 24-h EE during energy balance, fasting and four different overfeeding diets with 200% energy requirements was measured in a metabolic chamber in 37 subjects with normal glucose regulation while they resided on our clinical research unit. Diets were given for 24-h each and included: 1) low-protein (3%), 2) standard (50% carbohydrate, 20% protein), 3) high-fat (60%), and 4) high-carbohydrate (75%). Participants returned for follow-up 6-months after the initial measures. The decrease in 24-h EE during fasting and the increase with overfeeding were correlated. A larger reduction in EE during fasting, a smaller EE response to low-protein overfeeding and a larger response to high-carbohydrate overfeeding all correlated with weight gain. The association of the fasting EE response with weight change was not independent from that of low-protein in a multivariate model. We identified two independent propensities associated with weight gain: a predilection for conserving energy during caloric and protein deprivation, and a profligate response to large amounts of carbohydrates.
Authors: Scruggs BS, Gilchrist DA, Nechaev S, Muse GW, Burkholder A, Fargo DC, Adelman K
Journal: Mol Cell. 2015 Jun 18;58(6):1101-12. doi: 10.1016/j.molcel.2015.04.006. Epub 2015 May 28.
Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding, and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression.
Authors: Piaggi P, Thearle MS, Krakoff J, Votruba SB
Journal: J Clin Endocrinol Metab. 2015 Jun 18:jc20152164. [Epub ahead of print]
CONTEXT: Body fat free mass (FFM), energy expenditure (EE) and respiratory quotient (RQ) are known predictors of daily food intake. As FFM largely determines EE, it is unclear whether body composition per se or the underlying metabolism drives dietary intake.
OBJECTIVE: To test whether 24-h measures of EE and RQ and their components influence ad libitum food intake independently of FFM.
DESIGN AND PARTICIPANTS: One-hundred-seven healthy individuals (62M/45F, 84 Native Americans/23 Whites; age: 33±8 yrs.; BMI: 33±8 kg/m2; body fat: 31±8%) had 24-h measures of EE in a whole-room indirect calorimeter during energy balance, followed by three days of ad libitum food intake using computerized vending machine systems. Body composition was estimated by DXA.
MAIN OUTCOME MEASURES: FFM, 24-h EE, RQ, spontaneous physical activity (SPA), sleeping EE (SMR), "awake and fed" thermogenesis (AFT) and ad libitum food intake (INTAKE).
RESULTS: Higher 24-h RQ (P<0.001, partial R2=16%) and EE (P=0.01, partial R2=7%), but not FFM (P=0.65), were independent predictors of INTAKE. Mediation analysis demonstrated that 24-h EE is responsible for 80% of the FFM effect on INTAKE (44.5±16.9 kcal ingested per kg FFM, P=0.01), whereas the unique effect due to solely FFM was negligible (10.6±23.2, P=0.65). SPA (r=0.33, P=0.001), but not SMR (P=0.71), positively predicted INTAKE, while higher AFT determined greater INTAKE only in subjects with a BMI≤29 kg/m2 (r=0.44, P=0.01).
CONCLUSIONS: EE and RQ, rather than FFM, independently determine INTAKE, suggesting that competitive energy-sensing mechanisms driven by the preferential macronutrient oxidation and total energy demands may regulate food intake.
Authors: Xiao Y, Thoresen DT, Williams JS, Wang C, Perna J, Petrova R, Brownell I
Journals: Proc Natl Acad Sci U S A. 2015 Jun 9;112(23):7195-200. doi: 10.1073/pnas.1504177112. Epub 2015 May 26
The touch dome is a highly patterned mechanosensory structure in the epidermis composed of specialized keratinocytes in juxtaposition with innervated Merkel cells. The touch dome epithelium is maintained by tissue-specific stem cells, but the signals that regulate the touch dome are not known. We identify touch dome stem cells that are unique among epidermal cells in their activated Hedgehog signaling and ability to maintain the touch dome as a distinct lineage compartment. Skin denervation reveals that renewal of touch dome stem cells requires a perineural microenvironment, and deleting Sonic hedgehog (Shh) in neurons or Smoothened in the epidermis demonstrates that Shh is an essential niche factor that maintains touch dome stem cells. Up-regulation of Hedgehog signaling results in neoplastic expansion of touch dome keratinocytes but no Merkel cell neoplasia. These findings demonstrate that nerve-derived Shh is a critical regulator of lineage-specific stem cells that maintain specialized sensory compartments in the epidermis.
Authors: Wang XF, Bi GH, He Y, Yang HJ, Gao JT, Okunola-Bakare OM, Slack RD, Gardner EL, Xi ZX, Newman AH
Journal: Neuropsychopharmacology. 2015 Jun;40(7):1762-71. doi: 10.1038/npp.2015.24
(±)-Modafinil (MOD) is used clinically for the treatment of sleep disorders and has been investigated as a potential medication for the treatment of psychostimulant addiction. However, the therapeutic efficacy of (±)-MOD for addiction is inconclusive. Herein we used animal models of self-administration and in vivo microdialysis to study the pharmacological actions of R-modafinil (R-MOD) and S-modafinil (S-MOD) on nicotine-taking and nicotine-seeking behavior, and mechanisms underlying such actions. We found that R-MOD is more potent and effective than S-MOD in attenuating nicotine self-administration in Long-Evans rats. As Long-Evans rats did not show a robust reinstatement response to nicotine, we used alcohol-preferring rats (P-rats) that display much higher reinstatement responses to nicotine than Long-Evans rats. We found that R-MOD significantly inhibited intravenous nicotine self-administration, nicotine-induced reinstatement, and nicotine-associated cue-induced drug-seeking behavior in P-rats. R-MOD alone neither sustained self-administration in P-rats previously self-administering nicotine nor reinstated extinguished nicotine-seeking behavior. The in vivo brain microdialysis assays demonstrated that R-MOD alone produced a slow-onset moderate increase in extracellular DA. Pretreatment with R-MOD dose-dependently blocked nicotine-induced dopamine (DA) release in the nucleus accumbens (NAc) in both naive and nicotine self-administrating rats, suggesting a DA-dependent mechanism underlying mitigation of nicotine's effects. In conclusion, the present findings support further investigation of R-MOD for treatment of nicotine dependence in humans.