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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:

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.

Authors: Davis FM, Janoshazi A, Janardhan KS, Steinckwich N, D'Agostin DM, Petranka JG, Desai PN, Roberts-Thomson SJ, Bird GS, Tucker DK, Fenton SE, Feske S, Monteith GR, Putney JW Jr

Journal: Proc Natl Acad Sci U S A. 2015 May 5;112(18):5827-32. doi: 10.1073/pnas.1502264112. Epub 2015 Apr 20.

The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca(2+) channel subunit, Orai1, is required for both optimal Ca(2+) transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca(2+) in mammary myoepithelial cells followed by slow, irregular Ca(2+) oscillations. These oscillations, and not the initial Ca(2+) transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca(2+) is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca(2+) oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Authors: Alonso A, Huang X, Mosley TH, Heiss G, Chen H.

Journal: Ann Neurol. 2015 May;77(5):877-83. doi: 10.1002/ana.24393. Epub 2015 Mar 27.

OBJECTIVE: Autonomic dysfunction frequently occurs in the context of Parkinson disease (PD) and may precede onset of motor symptoms. Limited data exist on the prospective association of heart rate variability (HRV), a marker of autonomic function, with PD risk.

METHODS: We included 12,162 participants of the Atherosclerosis Risk in Communities study, a community-based cohort, without a diagnosis of PD at baseline (1987-1989) and with available HRV data (mean age = 54 years, 57% women). A 2-minute electrocardiogram was used to measure HRV. Incident PD was identified through 2008 from multiple sources, and adjudicated. Multivariable Cox models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of PD by quartiles of HRV measurements.

RESULTS: During a mean follow-up of 18 years, we identified 78 incident PD cases. Lower values of the root mean square of successive differences in normal-to-normal R-R intervals (rMSSD) and standard deviation of normal-to-normal R-R intervals (SDNN), markers of parasympathetic activity and total variability, respectively, were associated with higher PD risk during follow-up. In multivariate models, the HR (95% CI) of PD in the bottom quartiles of rMSSD and SDNN compared to the top quartiles were 2.1 (1.0-4.3) and 2.9 (1.4-6.1), respectively. Other measures of cardiac autonomic function, including mean R-R interval and frequency-domain measurements, were not associated with PD risk.

INTERPRETATION: In this prospective cohort, decreased HRV was associated with an increased risk of PD. Assessment of cardiac autonomic function may help identify individuals at risk for PD. Ann Neurol 2015;77:877-883.

Authors: Weinberg CR, Zaykin D

Journal: J Natl Cancer Inst. 2015 May 8;107(7). pii: djv125. doi: 10.1093/jnci/djv125. Print 2015 Jul.

A recent study reports that the log lifetime incidence rate across a selection of 31 cancer types is highly correlated with the log of the estimated tissue-specific lifetime number of stem cell divisions. This observation, which underscores the importance of errors in DNA replication, has been viewed as implying that most cancers arise through unavoidable bad luck, leading to the suggestion that research efforts should focus on early detection, rather than etiology or prevention. We argue that three statistical issues can, if ignored, lead analysts to incorrect conclusions. Statistics for traffic fatalities across the United States provide an example to demonstrate those inferential pitfalls. While the contribution of random cellular events to disease is often underappreciated, the role of chance is necessarily difficult to quantify. The conclusion that most cases of cancer are fundamentally unpreventable because they are the result of chance is unwarranted.

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