Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state.

MeCP2 is a nuclear protein with an affinity for methylated DNA that can recruit histone deacetylases. Deficiency or excess of MeCP2 causes severe neurological problems, suggesting that the number of molecules per cell must be precisely regulated. We quantified MeCP2 in neuronal nuclei and found that it is nearly as abundant as the histone octamer. Despite this high abundance, MeCP2 associates preferentially with methylated regions, and high-throughput sequencing showed that its genome-wide binding tracks methyl-CpG density. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. Neither change is detectable in glia, where MeCP2 occurs at lower levels. The mutant brain also shows elevated transcription of repetitive elements. Our data argue that MeCP2 may not act as a gene-specific transcriptional repressor in neurons, but might instead dampen transcriptional noise genome-wide in a DNA methylation-dependent manner.

Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome.

The possibility that alterations in DNA methylation are mechanistic drivers of development, aging and susceptibility to disease is widely acknowledged, but evidence remains patchy or inconclusive. Of particular interest in this regard is the brain, where it has been reported that DNA methylation impacts on neuronal activity, learning and memory, drug addiction and neurodegeneration. Until recently, however, little was known about the 'landscape' of the human brain methylome. Here we assay 1.9 million CpGs in each of 43 brain samples representing different individuals and brain regions. The cerebellum was a consistent outlier compared to all other regions, and showed over 16 000 differentially methylated regions (DMRs). Unexpectedly, the sequence characteristics of hypo- and hypermethylated domains in cerebellum were distinct. In contrast, very few DMRs distinguished regions of the cortex, limbic system and brain stem. Inter-individual DMRs were readily detectable in these regions. These results lead to the surprising conclusion that, with the exception of cerebellum, DNA methylation patterns are more homogeneous between different brain regions from the same individual, than they are for a single brain region between different individuals. This finding suggests that DNA sequence composition, not developmental status, is the principal determinant of the human brain DNA methylome.

CpG islands influence chromatin structure via the CpG-binding protein Cfp1.

CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides. Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity. In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro. Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins.

ChIP-seq and transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi and Typhimurium reveals accessory genes implicated in host colonization.

OmpR is a multifunctional DNA binding regulator with orthologues in many enteric bacteria that exhibits classical regulator activity as well as nucleoid-associated protein-like characteristics. In the enteric pathogen Salmonella enterica, using chromatin immunoprecipitation of OmpR:FLAG and nucleotide sequencing, 43 putative OmpR binding sites were identified in S. enterica serovar Typhi, 22 of which were associated with OmpR-regulated genes. Mutation of a sequence motif (TGTWACAW) that was associated with the putative OmpR binding sites abrogated binding of OmpR:6×His to the tviA upstream region. A core set of 31 orthologous genes were found to exhibit OmpR-dependent expression in both S. Typhi and S. Typhimurium. S. Typhimurium-encoded orthologues of two divergently transcribed OmpR-regulated operons (SL1068-71 and SL1066-67) had a putative OmpR binding site in the inter-operon region in S. Typhi, and were characterized using in vitro and in vivo assays. These operons are widely distributed within S. enterica but absent from the closely related Escherichia coli. SL1066 and SL1067 were required for growth on N-acetylmuramic acid as a sole carbon source. SL1068-71 exhibited sequence similarity to sialic acid uptake systems and contributed to colonization of the ileum and caecum in the streptomycin-pretreated mouse model of colitis.

FRT-seq: amplification-free, strand-specific transcriptome sequencing.

We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A)(+) RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Orphan CpG islands identify numerous conserved promoters in the mammalian genome.

CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are "orphans" that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development.

Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.

Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are closely related Gram-negative beta-proteobacteria that colonize the respiratory tracts of mammals. B. pertussis is a strict human pathogen of recent evolutionary origin and is the primary etiologic agent of whooping cough. B. parapertussis can also cause whooping cough, and B. bronchiseptica causes chronic respiratory infections in a wide range of animals. We sequenced the genomes of B. bronchiseptica RB50 (5,338,400 bp; 5,007 predicted genes), B. parapertussis 12822 (4,773,551 bp; 4,404 genes) and B. pertussis Tohama I (4,086,186 bp; 3,816 genes). Our analysis indicates that B. parapertussis and B. pertussis are independent derivatives of B. bronchiseptica-like ancestors. During the evolution of these two host-restricted species there was large-scale gene loss and inactivation; host adaptation seems to be a consequence of loss, not gain, of function, and differences in virulence may be related to loss of regulatory or control functions.

Strained cyclophane macrocycles: impact of progressive ring size reduction on synthesis and structure.

The synthesis, X-ray crystal structures, and calculated strain energies are reported for a homologous series of 11- to 14-membered drug-like cyclophane macrocycles, representing an unusual region of chemical space that can be difficult to access synthetically. The ratio of macrocycle to dimer, generated via a copper catalyzed azide-alkyne cycloaddition macrocyclization in flow at elevated temperature, could be rationalized in terms of the strain energy in the macrocyclic product. The progressive increase in strain resulting from reduction in macrocycle ring size, or the introduction of additional conformational constraints, results in marked deviations from typical geometries. These strained cyclophane macrocyclic systems provide access to spatial orientations of functionality that would not be readily available in unstrained or acyclic analogs. The most strained system prepared represents the first report of an 11-membered cyclophane containing a 1,4-disubstituted 1,2,3-triazole ring and establishes a limit to the ring strain that can be generated using this macrocycle synthesis methodology.

Macrocyclizations for medicinal chemistry: synthesis of druglike macrocycles by high-concentration Ullmann coupling.

Conditions have been identified for the efficient Ullmann macrocyclization of phenol and imidazole nucleophiles with aryl iodides at high reaction concentrations of up to 100 mM and using 5-10 mol % loading of an inexpensive copper catalyst. A range of substitution patterns and ring sizes are tolerated, and the method has been exemplified by the synthesis of a set of druglike macrocycles.

Biaryl-bridged macrocyclic peptides: conformational constraint via carbogenic fusion of natural amino acid side chains.

A general method for constraining peptide conformations via linkage of aromatic sidechains has been developed. Macrocyclization of suitably functionalized tri-, tetra- and pentapeptides via Suzuki-Miyaura cross-coupling has been used to generate side chain to side chain, biaryl-bridged 14- to 21-membered macrocyclic peptides. Biaryl bridges possessing three different configurations, meta-meta, meta-ortho, and ortho-meta, were systematically explored through regiochemical variation of the aryl halide and aryl boronate coupling partners, allowing fine-tuning of the resultant macrocycle conformation. Suzuki-Miyaura macrocyclizations were successfully achieved both in solution and on solid phase for all three sizes of peptide. This approach constitutes a means of constraining peptide conformation via direct carbogenic fusion of side chains of naturally occurring amino acids such as phenylalanine and tyrosine, and so is complementary to strategies involving non-natural, for example, hydrocarbon, bridges.

A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi.

High-density, strand-specific cDNA sequencing (ssRNA-seq) was used to analyze the transcriptome of Salmonella enterica serovar Typhi (S. Typhi). By mapping sequence data to the entire S. Typhi genome, we analyzed the transcriptome in a strand-specific manner and further defined transcribed regions encoded within prophages, pseudogenes, previously un-annotated, and 3'- or 5'-untranslated regions (UTR). An additional 40 novel candidate non-coding RNAs were identified beyond those previously annotated. Proteomic analysis was combined with transcriptome data to confirm and refine the annotation of a number of hpothetical genes. ssRNA-seq was also combined with microarray and proteome analysis to further define the S. Typhi OmpR regulon and identify novel OmpR regulated transcripts. Thus, ssRNA-seq provides a novel and powerful approach to the characterization of the bacterial transcriptome.

Identification of Aluminium Powder Properties for Modelling Free Air Explosions.

Aluminium is a component in many energetic formulations. Therefore, its combustion is one of the main thermochemical processes that govern the output from the energetics. Modelling aluminium combustion is a challenging task because the process is highly complex and difficult to measure. Here, tests of aluminium powder were conducted in an effort to isolate the burning of the aluminium and to determine an adequate representation of this process. Charges of 100 g and 500 g were tested, and the size of the Al/air cloud and the ratio of components in the Al/air mixture were determined, which has not been published previously. This information was used to assess the validity of the assumption that the detonation of the mixture was representative of the event. Parameters for the Jones-Wilkins-Lee equation of state for the explosive mixture and detonation products were defined. Simulations of the tests were performed, and the results were consistent with the field test data, indicating that detonation occurred when there was a mixture of 70-75% Al and 25-30% air by mass.

Comparison of diffusion coefficients for matched pairs of macrocyclic and linear molecules over a drug-like molecular weight range.

The diffusion coefficients of a series of closely matched pairs of macrocyclic and linear molecules have been compared using NMR spectroscopy. The macrocyclic series was designed both to overlap with and extend beyond the molecular weight range typically employed for drug-like molecules. The linear molecules each represent a carbogenic fission of their macrocyclic counterparts, designed to minimize differences in functionality and physicochemical properties. Each series of molecules was prepared using copper catalyzed azide-alkyne cycloaddition (CuAAC) reactions conducted in a flow using a copper tube. The macrocyclic series exhibited consistently higher diffusion across the entire molecular weight range studied. The fold difference in diffusion coefficients between the macrocyclic and linear analogues appeared to be independent of either solvent viscosity or dielectric environment.

Efficient access to new chemical space through flow--construction of druglike macrocycles through copper-surface-catalyzed azide-alkyne cycloaddition reactions.

A series of 12- to 22-membered macrocycles, with druglike functionality and properties, have been generated by using a simple and efficient copper-catalyzed azide-acetylene cycloaddition reaction, conducted in flow in high-temperature copper tubing, under environmentally friendly conditions. The triazole-containing macrocycles have been generated in up to 90 % yield in a 5 min reaction, without resorting to the high-dilution conditions typical of macrocyclization reactions. This approach represents a very efficient method for constructing this important class of molecules, in terms of yield, concentration, and environmental considerations.

Ketone body modulation of ligand-gated ion channels.

Ketogenesis is a metabolic process wherein ketone bodies are produced from the breakdown of fatty acids. In humans, fatty acid catabolism results in the production of acetyl-CoA which can then be used to synthesize three ketone bodies: acetoacetate, acetone, and ß-hydroxybutyrate. Ketogenesis occurs at a higher rate in situations of low blood glucose, such as during fasting, heavy alcohol consumption, and in situations of low insulin, as well as in individuals who follow a 'ketogenic diet' consisting of low carbohydrate and high fat intake. This diet has various therapeutic indications, including reduction of seizure likelihood in epileptic patients and alcohol withdrawal syndrome. However, the mechanisms underlying these therapeutic benefits are still unclear, with studies suggesting various mechanisms such as a shift in energy production in the brain, effects on neurotransmitter production, or effects on various protein targets. Two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes was used to investigate the actions of ketone bodies on three ionotropic receptors: GABAA, glycine, and NMDA receptors. While physiologically-relevant concentrations of acetone have little effect on inhibitory GABA or glycine receptors, ß-hydroxybutyrate inhibits the effects of agonists of these receptors at concentrations achieved in vivo. Additionally, both acetone and ß-hydroxybutyrate act as inhibitors of glutamate at the excitatory NMDA receptor. Due to the role of hyperexcitability in the pathogenesis of epilepsy and alcohol withdrawal, the inhibitory actions of acetone and ß-hydroxybutyrate at NMDA receptors may underlie the therapeutic benefit of a ketogenic diet for these disorders.

Mortality and morbidity of major congenital heart disease related to general prenatal screening for malformations.

BACKGROUND: Major congenital heart diseases (CHD) often demand intervention in the neonatal period. Prenatal diagnosis may improve mortality by eliminating the diagnostic delay; however, there is controversy concerning its true effect. We aimed to evaluate the effect of general prenatal screening on prognosis by comparing a period without general prenatal screening to a period with general prenatal screening. METHODS: We conducted a nationwide retrospective study including live born children and terminated fetuses diagnosed with major CHD. Prenatal screening was recommended only in high risk pregnancies between 1996 and 2004, whereas general prenatal screening was recommended between 2005 and 2013. We assessed the influence of general prenatal screening on all-cause mortality, cardiac death, preoperative and postoperative 30-day mortality and complication rate. RESULTS: 1-year mortality decreased over both periods, but the decrease was greater in the screening period (Odds ratio 0.92 (CI 0.83-1.00), p = 0.047). Prenatal detection of major CHD was associated with cardiac death in the period without general screening (Hazard Ratio 2.40 (CI 1.72-3.33), p < 0.001), whereas there was no significant association once general screening was implemented. Similarly, the association between prenatal diagnosis and pre- and postoperative mortality found in the period without general screening was insignificant after the implementation of general screening. CONCLUSION: Mortality in major CHD decreased throughout the study, especially in the period with general prenatal screening. However, comparing a prenatally diagnosed group with a postnatally diagnosed group is vulnerable to selection bias and proper interpretation is difficult.

Terrorism and hazardous material trucking: promoting perceived collective efficacy for terrorism prevention.

Hazardous-material trucking has recently been identified as an area of high potential risk for terrorism. Some recent theory and case study papers have argued for the importance of collective efficacy to disaster-response, terrorism prevention, and other rare-but-risky events. Therefore, a study based on the collective efficacy literature was done to test an intervention for increasing perceived collective efficacy for terrorism prevention among Canadian hazardous-material truck drivers. Results supported the impact of the intervention in increasing perceived efficacy for terrorism prevention. Implications for theory, research, and application are discussed.

Live-Born Major Congenital Heart Disease in Denmark: Incidence, Detection Rate, and Termination of Pregnancy Rate From 1996 to 2013.

Importance: The occurrence of major congenital heart disease (CHD) is affected by several variables. Determining the development of the true incidence is critical to the establishment of proper treatment of these patients. Objective: To evaluate time trends in incidence, detection rate, and termination of pregnancy (TOP) rate of major CHD in fetuses in Denmark and assess the influence of the introduction of general prenatal screening in 2004. Design, Setting, and Participants: Nationwide, population-based, retrospective observational study in Denmark from 1996 to 2013 that included a consecutive sample of 14 688 live-born children and terminated fetuses diagnosed as having CHD. Patient records on TOP and children with major CHD were reviewed to validate the diagnoses. Major CHD included univentricular heart, transposition of the great arteries, congenitally corrected transposition of the great arteries, truncus arteriosus, interrupted aortic arch, atrioventricular septal defects, double outlet right ventricle, coarctatio of the aorta, Ebstein anomaly, pulmonary atresia with ventricular septal defect, pulmonary atresia with intact ventricular septum, and tetralogy of Fallot. Data were analyzed between January 2017 and March 2018. Main Outcomes and Measures: Temporal changes in incidence, detection rate, and TOP of major CHD. Results: Of 14 688 children and fetuses diagnosed with CHD, 2695 (18.4%; 95% CI, 17.8-19.1) had major CHD. A total of 7131 boys (1304 with major CHD) and 6926 girls (920 with major CHD) were included, with a median age of 11 years (interquartile range, 6-15 years). During the study period, the live-birth incidence of CHD was constant at 1.22% (95% CI, 1.18-1.26), whereas it decreased for major CHD. When including TOP, the incidence of major CHD did not change over time. The detection rate of major CHD increased from 4.5% (95% CI, 1.2-7.8) to 71.0% (95% CI, 63.3-78.7) (P < .001). At the end of the study, all cases of double outlet right ventricle, Ebstein anomaly, congenitally corrected transposition of the great arteries, and pulmonary atresia with ventricular septal defect were detected prenatally, whereas coarctation of the aorta had the lowest detection rate (21.7%; 95% CI, 3.5-40.0). The TOP rate increased from 0.6% (95% CI, -0.6 to 1.9) to 39.1% (95% CI, 30.9-47.4) (P < .001) among all major CHD. For prenatally diagnosed major CHD, 57.8% of cases were terminated and the proportion did not change significantly throughout the study. Diagnoses leading to TOP included all major CHD diagnoses. Conclusions and Relevance: Detection rates of major CHD improved during the study. This has led to increased TOP rates, with a subsequent 39% decrease in the live-birth incidence of major CHD.

Age differences in feedback reactions: The roles of employee feedback orientation on social awareness and utility.

Organizations worldwide are currently experiencing shifts in the age composition of their workforces. The workforce is aging and becoming increasingly age-diverse, suggesting that organizational researchers and practitioners need to better understand how age differences may manifest in the workplace and the implications for human resource practice. Integrating socioemotional selectivity theory with the performance feedback literature and using a time-lagged design, the current study examined age differences in moderating the relationships between the characteristics of performance feedback and employee reactions to the feedback event. The results suggest that older workers had higher levels of feedback orientation on social awareness, but lower levels of feedback orientation on utility than younger workers. Furthermore, the positive associations between favorability of feedback and feedback delivery and feedback reactions were stronger for older workers than for younger workers, whereas the positive association between feedback quality and feedback reactions was stronger for younger workers than for older workers. Finally, the current study revealed that age-related differences in employee feedback orientation could explain the different patterns of relationships between feedback characteristics and feedback reactions across older and younger workers. These findings have both theoretical and practical implications for building theory about workplace aging and improving ways that performance feedback is managed across employees from diverse age groups.