Methyl CpG Binding Domain Ultra-Sequencing: a novel method for identifying inter-individual and cell-type-specific variation in DNA methylation

Experience-dependent changes in DNA methylation can exert profound effects on neuronal function and behaviour. A single learning event can induce a variety of DNA modifications within the neuronal genome, some of which may be common to all individuals experiencing the event, whereas others may occur in a subset of individuals. Variations in experience-induced DNA methylation may subsequently confer increased vulnerability or resilience to the development of neuropsychiatric disorders. However, the detection of experience-dependent changes in DNA methylation in the brain has been hindered by the interrogation of heterogeneous cell populations, regional differences in epigenetic states and the use of pooled tissue obtained from multiple individuals. Methyl CpG Binding Domain Ultra-Sequencing (MBD Ultra-Seq) overcomes current limitations on genome-wide epigenetic profiling by incorporating fluorescence-activated cell sorting and sample-specific barcoding to examine cell-type-specific CpG methylation in discrete brain regions of individuals. We demonstrate the value of this method by characterizing differences in 5-methylcytosine (5mC) in neurons and non-neurons of the ventromedial prefrontal cortex of individual adult C57BL/6 mice, using as little as 50 ng of genomic DNA per sample. We find that the neuronal methylome is characterized by greater CpG methylation as well as the enrichment of 5mC within intergenic loci. In conclusion, MBD Ultra-Seq is a robust method for detecting DNA methylation in neurons derived from discrete brain regions of individual animals. This protocol will facilitate the detection of experience-dependent changes in DNA methylation in a variety of behavioural paradigms and help identify aberrant experience-induced DNA methylation that may underlie risk and resiliency to neuropsychiatric disease

Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation

5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation

Hyperthermia Induces Functional and Molecular Modifications in Cardiac, Smooth and Skeletal Muscle Cells

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionHyperthermia is used for the treatment of a number of diseases, including muscle injuries, inflammations, tendinitis, and osteoarticular disorder. More recently, hyperthermia has been used as an adjuvant in cancer treatment. Only two studies have shown that hyperthermia leads to hypertrophy in in-vitro models of cardiac and skeletal muscle cells. Functional, biochemical and molecular mechanisms of hyperthermia-induced hypertrophy in muscles remain largely undiscovered. We investigated the effects of mild heat shock (HS) on C2C12 skeletal, HL-1 cardiac and AR-75 smooth muscle cells. Mild HS (20 min 43ºC) induced increases in the cell area in all muscle cells tested. C2C12 cells are a well-accepted model of skeletal muscle fibers, and were selected for complementary studies. First, to biochemically confirm an increase in protein synthesis we measured and found an increase of ~6% in total protein content 24 hrs after HS. Second, we examined potential modifications in calcium (Ca) homeostasis regulation by measuring intracellular Ca. We detected a lower resting level of intracellular Ca and smaller and longer caffeine-induced Ca transients in C2C12 muscle cells 24 hrs after HS. Next, to search for molecular mechanisms involved with HS-induced hypertrophy and calcium homeostasis modifications, mRNA from C2C12 muscle cells was analyzed at different time points after HS (0, 1, 2, and 24 hrs). We used an ABI Step One Plus RT2 PCR Array System and a custom-built 96 gene array. We report for the first time that the expression of key heat-shock, hypertrophy/ metabolic, and Ca+2 signaling genes were altered after HS. Hsp70 and Hsp72 genes were highly expressed (211-1829 fold change) after HS. Also, Myh7 (MHC-I), Myh6, Srf, Ppp3r1 and Pck1 were up-regulated by 2-6 fold change compared with control cells.. Furthermore, a reduction in the expression of RyR and Trdn genes was observed (2- 3.6 fold change) with an associated increase in the expression of IP3R genes (2-4 fold change). These results indicate that hyperthermia modulates not only heat-shock related and hypertrophy genes, but also genes involved with metabolism, apoptosis repression, calcium homeostasis and signaling, and cell homeostasis. Our studies offer an initial exploration of the functional, biochemical and molecular mechanisms that may help explain the beneficially adaptive effects of hyperthermia on muscle function. Our studies shall also prove useful for the refinement of a specific device (EM-Stim) to be employed for the treatment of muscle and bone diseases (See poster by Hatem et al). Importantly, our studies have potential translational applications. By learning how to more precisely use hyperthermia to control specific genes that can improve or treat muscle injuries, musculoskeletal, and cardiovascular diseases, the ensuing benefits shall be unmistakable. Our short and long-term goals are: i) optimize our protocols; ii) test HS in animal models; iii) manipulate expression of promising genes of interest in vitro and in in-vivo animal models; iv) initiate clinical studies to fully translate from the bench to the bed-side

Observation of the 1S–2S transition in trapped antihydrogen

International audienceThe spectrum of the hydrogen atom has played a central part in fundamental physics in the past 200 years. Historical examples of its significance include the wavelength measurements of absorption lines in the solar spectrum by Fraunhofer, the identification of transition lines by Balmer, Lyman et al., the empirical description of allowed wavelengths by Rydberg, the quantum model of Bohr, the capability of quantum electrodynamics to precisely predict transition frequencies, and modern measurements of the 1S–2S transition by Hänsch to a precision of a few parts in 1015. Recently, we have achieved the technological advances to allow us to focus on antihydrogen—the antimatter equivalent of hydrogen. The Standard Model predicts that there should have been equal amounts of matter and antimatter in the primordial Universe after the Big Bang, but today’s Universe is observed to consist almost entirely of ordinary matter. This motivates physicists to carefully study antimatter, to see if there is a small asymmetry in the laws of physics that govern the two types of matter. In particular, the CPT (charge conjugation, parity reversal, time reversal) Theorem, a cornerstone of the Standard Model, requires that hydrogen and antihydrogen have the same spectrum. Here we report the observation of the 1S–2S transition in magnetically trapped atoms of antihydrogen in the ALPHA-2 apparatus at CERN. We determine that the frequency of the transition, driven by two photons from a laser at 243 nm, is consistent with that expected for hydrogen in the same environment. This laser excitation of a quantum state of an atom of antimatter represents a highly precise measurement performed on an anti-atom. Our result is consistent with CPT invariance at a relative precision of $\sim 2 × 10^{-10}$

Why place matters in residential care: the mediating role of place attachment in the relation between adolescents’ rights and psychological well-being

Little evidence exists on the relationship between rights’ perceptions and well-being outcomes during the adolescence, and particularly in care, as well as on the mediating role of place attachment. Young people in residential care are psychologically and socially vulnerable, showing greater difficulties than their peers do in the family. Youth’s rights fulfilment in residential care may positively affect their psychological functioning together with positive attachments to this place. A sample of 365 adolescents in residential care settings (M = 14.71, SD = 1.81) completed a set of self-reported measures, specifically, the Rights perceptions scale, the Place attachment scale and Scales of psychological well-being. Results revealed significant mediating effects of place attachment (Global scale and subscales of Friends Bonding and Place Dependence) on the relationship between Participation and Protection rights in residential care and Psychological well-being (Positive Relations with others, Personal Growth and Self-Acceptance). The positive role of rights fulfilment in residential care, specifically participation opportunities, as well as the role of youth’s attachment to the care setting are discussed based on previous evidence and theoretical assumptions. A set of practical implications is described.info:eu-repo/semantics/acceptedVersio

Sustained Na⁺/H⁺ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na +/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1-5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1-24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H + homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI. © 2014 Cengiz et al

Observation of the 1S–2P Lyman-α transition in antihydrogen

International audienceIn 1906, Theodore Lyman discovered his eponymous series of transitions in the extreme-ultraviolet region of the atomic hydrogen spectrum 1$^{,}$2 . The patterns in the hydrogen spectrum helped to establish the emerging theory of quantum mechanics, which we now know governs the world at the atomic scale. Since then, studies involving the Lyman-α line—the 1S–2P transition at a wavelength of 121.6 nanometres—have played an important part in physics and astronomy, as one of the most fundamental atomic transitions in the Universe. For example, this transition has long been used by astronomers studying the intergalactic medium and testing cosmological models via the so-called ‘Lyman-α forest’ 3 of absorption lines at different redshifts. Here we report the observation of the Lyman-α transition in the antihydrogen atom, the antimatter counterpart of hydrogen. Using narrow-line-width, nanosecond-pulsed laser radiation, the 1S–2P transition was excited in magnetically trapped antihydrogen. The transition frequency at a field of 1.033 tesla was determined to be 2,466,051.7 ± 0.12 gigahertz (1σ uncertainty) and agrees with the prediction for hydrogen to a precision of 5 × 10$^{−8}$. Comparisons of the properties of antihydrogen with those of its well-studied matter equivalent allow precision tests of fundamental symmetries between matter and antimatter. Alongside the ground-state hyperfine 4$^{,}$5 and 1S–2S transitions 6$^{,}$7 recently observed in antihydrogen, the Lyman-α transition will permit laser cooling of antihydrogen 8$^{,}$9 , thus providing a cold and dense sample of anti-atoms for precision spectroscopy and gravity measurements 10 . In addition to the observation of this fundamental transition, this work represents both a decisive technological step towards laser cooling of antihydrogen, and the extension of antimatter spectroscopy to quantum states possessing orbital angular momentum

Observation of the hyperfine spectrum of antihydrogen

The observation of hyperfine structure in atomic hydrogen by Rabi and co-workers and the measurement of the zero-field ground-state splitting at the level of seven parts in 1013 are important achievements of mid-twentieth-century physics. The work that led to these achievements also provided the first evidence for the anomalous magnetic moment of the electron, inspired Schwinger’s relativistic theory of quantum electrodynamics, and gave rise to the hydrogen maser, which is a critical component of modern navigation, geo-positioning and very-long-baseline interferometry systems. Research at the Antiproton Decelerator at CERN by the ALPHA collaboration extends these enquiries into the antimatter sector. Recently, tools have been developed that enable studies of the hyperfine structure of antihydrogen—the antimatter counterpart of hydrogen. The goal of such studies is to search for any differences that might exist between this archetypal pair of atoms, and thereby to test the fundamental principles on which quantum field theory is constructed. Magnetic trapping of antihydrogen atoms provides a means of studying them by combining electromagnetic interaction with detection techniques that are unique to antimatter. Here we report the results of a microwave spectroscopy experiment in which we probe the response of antihydrogen over a controlled range of frequencies. The data reveal clear and distinct signatures of two allowed transitions, from which we obtain a direct, magnetic-field-independent measurement of the hyperfine splitting. From a set of trials involving 194 detected atoms, we determine a splitting of 1,420.4 ± 0.5 megahertz, consistent with expectations for atomic hydrogen at the level of four parts in 104. This observation of the detailed behaviour of a quantum transition in an atom of antihydrogen exemplifies tests of fundamental symmetries such as charge–parity–time in antimatter, and the techniques developed here will enable more-precise such tests

Antihydrogen formation in low-energy antiproton collisions with excited-state positronium atoms

© 2018, Springer Nature Switzerland AG. The convergent close-coupling method is used to obtain cross sections for antihydrogen formation in low-energy antiproton collisions with positronium (Ps) atoms in specified initial excited states with principal quantum numbers ni= 5. The threshold behaviour as a function of the Ps kinetic energy, E, is consistent with the 1/E law expected from threshold theory for all initial states. We find that the increase in the cross sections is muted above ni= 3 and that here their scaling is roughly consistent with ni2, rather than the classically expected increase as ni4

BDNF Methylation and Maternal Brain Activity in a Violence-Related Sample

It is known that increased circulating glucocorticoids in the wake of excessive, chronic, repetitive stress increases anxiety and impairs Brain-Derived Neurotrophic Factor (BDNF) signaling. Recent studies of BDNF gene methylation in relation to maternal care have linked high BDNF methylation levels in the blood of adults to lower quality of received maternal care measured via self-report. Yet the specific mechanisms by which these phenomena occur remain to be established. The present study examines the link between methylation of the BDNF gene promoter region and patterns of neural activity that are associated with maternal response to stressful versus non-stressful child stimuli within a sample that includes mothers with interpersonal violence-related PTSD (IPV-PTSD). 46 mothers underwent fMRI. The contrast of neural activity when watching children-including their own-was then correlated to BDNF methylation. Consistent with the existing literature, the present study found that maternal BDNF methylation was associated with higher levels of maternal anxiety and greater childhood exposure to domestic violence. fMRI results showed a positive correlation of BDNF methylation with maternal brain activity in the anterior cingulate (ACC), and ventromedial prefrontal cortex (vmPFC), regions generally credited with a regulatory function toward brain areas that are generating emotions. Furthermore we found a negative correlation of BDNF methylation with the activity of the right hippocampus. Since our stimuli focus on stressful parenting conditions, these data suggest that the correlation between vmPFC/ACC activity and BDNF methylation may be linked to mothers who are at a disadvantage with respect to emotion regulation when facing stressful parenting situations. Overall, this study provides evidence that epigenetic signatures of stress-related genes can be linked to functional brain regions regulating parenting stress, thus advancing our understanding of mothers at risk for stress-related psychopathology