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Understanding the biologic role of N-6-methyladenosine (m(6)A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m(6)A in exons but very rarely in introns. The m(6)A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m(6)A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only similar to 10% of m(6)As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m(6)A in wild-type mouse stem cells is spliced the same in cells lacking the major m(6)A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m(6)As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m(6)A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m(6)A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval(1), juvenile interactions(2), opposite and same sex interactions(3,4,5), and territorial interactions(6). For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders(7,8,9), and more recently to understand mechanisms and evolution of vocal communication among vertebrates(10). Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds(11). The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal's preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Osteoporosis is characterized by low bone density, and osteopenia is responsible for 1.5 million fractures in the United States annually.(1) In order to identify regions of the genome which are likely to contain genes predisposing to osteopenia, we genotyped 149 members of seven large pedigrees having recurrence of low bone mineral density (BMD) with 330 DNA markers spread throughout the autosomal genome. Linkage analysis for this quantitative trait was carried out using spine and hip BMD values by the classical lod-score method using a genetic model with parameters estimated from the seven families. In addition, non parametric analysis was performed using the traditional Haseman-Elston approach in 74 independent sib pairs from the same pedigrees. The maximum lod score obtained by parametric analysis in all families combined was +2.08 (theta = 0.05) for the marker CD3D on chromosome 11q. All other combined lod scores from the parametric analysis were less than +1.90, the threshold for suggestive linkage. Non-parametric analysis suggested linkage of low BMD to chromosomes 1p36 (Z(max) = +3.51 for D1S450) and 2p23-24 (Z(max) = +2.07 for D2S149). Maximum multi-point lod scores for these regions were +2.29 and +2.25, respectively. A third region with associated lod scores above the threshold of suggestive linkage in both single-point and multi point non-parametric analysis was on chromosome 4qter (Z(max) = +2.95 for D4S1539 and Z(max) = +2.48 for D4S1554). Our data suggest the existence of multiple genes involved in controlling spine and hip BMD, and indicate several candidate regions for further screening in this and other independent samples.

Mud is a porous medium containing a high density of diverse microorganisms. It is out of equilibrium as the energy from a photon flux is dissipated by a cascade of biochemical reactions, mediated by the metabolisms of the constituent organisms. Despite its complexity, microbes in nature self-organize into simple reproducible patterns. We present two experiments in which the dynamics of natural mud coming to steady state are observed and modeled. In the first, the oxygen gradient produced by cyanobacteria in an imposed light gradient is measured. In the second, a thin front of oxygen-consuming microbes forms at the penetration depth of oxygen and moves with the changing oxygen gradient.

Vascular abnormalities and inflammation are found in many Alzheimer disease (AD) patients, but whether these changes play a causative role in AD is not clear. The factor XII (FXII)-initiated contact system can trigger both vascular pathology and inflammation and is activated in AD patients and AD mice. We have investigated the role of the contact system in AD pathogenesis. Cleavage of high-molecular-weight kininogen (HK), a marker for activation of the inflammatory arm of the contact system, is increased in a mouse model of AD, and this cleavage is temporally correlated with the onset of brain inflammation. Depletion of FXII in AD mice inhibited HK cleavage in plasma and reduced neuroinflammation, fibrinogen deposition, and neurodegeneration in the brain. Moreover, FXII-depleted AD mice showed better cognitive function than untreated AD mice. These results indicate that FXII-mediated contact system activation contributes to AD pathogenesis, and therefore this system may offer novel targets for AD treatment.

The mechanisms by which early chronic low-level lead (Pb) exposure disrupts the developing brain are not yet understood. Rodent models have provided promising results however behavioral tests sensitive to effects at lowest levels of exposure during development are needed. Preadolescent animals (N = 52) exposed to low and higher levels of Pb via lactation from birth to PND 28 completed the Object-in-Place Task of visual spatial and visual object memory retrieval (at PND 28). Generalized linear mixed models were used, controlling for sex and litter as a random effect. As compared with controls, global vertical exploratory behavior (rearing) markedly increased during memory retrieval. The findings suggested that early chronic Pb exposure altered the development of critical exploratory functions needed for learning and survival. Behaviors exhibited in novel spatial and novel object zone perimeters suggested that the Object-in-Place task is a valid measure of visual spatial and visual object memory in pre-adolescent C57BL/6J mice. Additional studies are needed to understand how early chronic low-level lead exposure disrupts the trajectory and possible linkages of critical exploratory and perceptual systems during development. (C) 2017 Elsevier Inc. All rights reserved.

The CRISPR (clustered regularly interspaced short palindromic repeats) mechanism allows bacteria to adaptively defend against phages by acquiring short genomic sequences (spacers) that target specific sequences in the viral genome. We propose a population dynamical model where immunity can be both acquired and lost. The model predicts regimes where bacterial and phage populations can co-exist, others where the populations exhibit damped oscillations, and still others where one population is driven to extinction. Our model considers two key parameters: (1) ease of acquisition and (2) spacer effectiveness in conferring immunity. Analytical calculations and numerical simulations show that if spacers differ mainly in ease of acquisition, or if the probability of acquiring them is sufficiently high, bacteria develop a diverse population of spacers. On the other hand, if spacers differ mainly in their effectiveness, their final distribution will be highly peaked, akin to a "winner-take-all" scenario, leading to a specialized spacer distribution. Bacteria can interpolate between these limiting behaviors by actively tuning their overall acquisition probability.

Opioid receptor antagonist naltrexone reduces alcohol consumption and relapse in both humans and rodents. This study investigated whether hypothalamic proopiomelanocortin (POMC) neurons (producing beta-endorphin and melanocortins) play a role in alcohol drinking behaviors. Both male and female mice with targeted deletion of two neuronal Pomc enhancers nPE1 and nPE2 (nPE-/-), resulting in hypothalamic-specific POMC deficiency, were studied in short-access (4-h/day) drinking-in-the-dark (DID, alcohol in one bottle, intermittent access (IA, 24-h cycles of alcohol access every other day, alcohol vs. water in a two-bottle choice) and alcohol deprivation effect (ADE) models. Wild-type nPE+/+ exposed to 1-week DID rapidly established stable alcohol drinking behavior with more intake in females, whereas nPE-/- mice of both sexes had less intake and less preference. Although nPE-/- showed less saccharin intake and preference than nPE+/+, there was no genotype difference in sucrose intake or preference in the DID paradigm. After 3-week IA, nPE+/+ gradually escalated to high alcohol intake and preference, with more intake in females, whereas nPE-/- showed less escalation. Pharmacological blockade of mu-opioid receptors with naltrexone reduced intake in nPE+/+ in a dose-dependent manner, but had blunted effects in nPE-/- of both sexes. When alcohol was presented again after 1-week abstinence from IA, nPE+/+ of both sexes displayed significant increases in alcohol intake (ADE or relapse-like drinking), with more pronounced ADE in females, whereas nPE-/- did not show ADE in either sex. Our results suggest that neuronal POMC is involved in modulation of alcohol 'binge' drinking, escalation and 'relapse', probably via hypothalamic-mediated mechanisms, with sex differences.

The persistent vegetative state (PVS) is one of the most iconic and misunderstood phrases in clinical neuroscience. Coined as a diagnostic category by Scottish neurosurgeon Bryan Jennett and American neurologist Fred Plum in 1972, the phrase vegetative first appeared in Aristotle's treatise On the Soul (circa mid-fourth century BCE). Aristotle influenced neuroscientists of the nineteenth and early-twentieth centuries, Xavier Bichat and Walter Timme, and informed their conceptions of the vegetative nervous system. Plum credits Bichat and Timme in his use of the phrase, thus putting the ancient and modern in dialogue. In addition to exploring Aristotle's definition of the vegetative in the original Greek, we put Aristotle in conversation with his contemporariesPlato and the Hippocraticsto better apprehend theories of mind and consciousness in antiquity. Utilizing the discipline of reception studies in classics scholarship, we demonstrate the importance of etymology and historical origin when considering modern medical nosology.

BACKGROUND Myocarditis is inflammation of the heart muscle that can follow various viral infections. Why children only rarely develop life-threatening acute viral myocarditis (AVM), given that the causal viral infections are common, is unknown. Genetic lesions might underlie such susceptibilities. Mouse genetic studies demonstrated that interferon (IFN)-alpha/beta immunity defects increased susceptibility to virus-induced myocarditis. Moreover, variations in human TLR3, a potent inducer of IFNs, were proposed to underlie AVM. OBJECTIVES This study sought to evaluate the hypothesis that human genetic factors may underlie AVM in previously healthy children. METHODS We tested the role of TLR3-IFN immunity using human induced pluripotent stem cell-derived cardiomyocytes. We then performed whole-exome sequencing of 42 unrelated children with acute myocarditis (AM), some with proven viral causes. RESULTS We found that TLR3-and STAT1-deficient cardiomyocytes were not more susceptible to Coxsackie virus B3 (CVB3) infection than control cells. Moreover, CVB3 did not induce IFN-alpha/beta and IFN-alpha/beta-stimulated genes in control cardiomyocytes. Finally, exogenous IFN-alpha did not substantially protect cardiomyocytes against CVB3. We did not observe a significant enrichment of rare variations in TLR3-or IFN-alpha/beta-related genes. Surprisingly, we found that homozygous but not heterozygous rare variants in genes associated with inherited cardiomyopathies were significantly enriched in AM-AVM patients compared with healthy individuals (p = 2.22E-03) or patients with other diseases (p = 1.08E-04). Seven of 42 patients (16.7%) carried rare biallelic (homozygous or compound heterozygous) nonsynonymous or splice-site variations in 6 cardiomyopathy-associated genes (BAG3, DSP, PKP2, RYR2, SCN5A, or TNNI3). CONCLUSIONS Previously silent recessive defects of the myocardium may predispose to acute heart failure presenting as AM, notably after common viral infections in children. (C) 2017 by the American College of Cardiology Foundation.