Characterization and Antimicrobial Activity of Spice Extract-Loaded Algal Microcomposites Based on the Microemulsion Technique.

In recent years, with the increase in knowledge and awareness, people's efforts to return to nature have increased in the field of medical science and cosmetics industry. Spices, sumac and coriander, grown and frequently used in Turkey, have different bioactive effects. Microalgae are preferred in the treatment of skin problems. The aim of this study was to synthesize algae microcomposites that were effective against bacterial infections, prepared based on microemulsion technique and loaded with spice extract. Microemulsion formulations were prepared by the titration method. Aqueous and ethanolic extractions of sumac/coriander were carried out using the ultrasonic-assisted extraction method. Twenty-four different algal microcomposites loaded with extracts were synthesized. The disk diffusion method was used to determine the antimicrobial activity. The DPPH free radical scavenging activities, the total phenolic content (TPSC), and the characteristics (FT-IR) of the microcomposites were investigated. In addition, the chemical contents of extracts were determined by the GC/MS method. Aqueous extracts of both sumac and coriander were highly effective against Escherichia coli (ES DII). The highest antimicrobial activity against Staphylococcus aureus (F6 III) was obtained with M9 (microcomposite containing ethanolic extract of sumac) and M15 (microcomposite containing aqueous extract of coriander). The highest TPSC value (6.025 mg GAE/gr) was detected in the aqueous extract of coriander. The DPPH radical scavenging activities of coriander extracts were lower than those of sumac extracts. It has been determined that the spices contain organic (propanoic, butanoic, malic and benzoic) acids and fatty (palmitic, oleic and myristic) acids. According to the results of FT-IR spectroscopy, microcomposites prepared with sumac and coriander extracts were successfully synthesized. The synthesized algae-based microcomposites have properties that could be in the green-labeled bio-based category.

Epigenetic-genetic chromatin footprinting identifies novel and subject-specific genes active in prefrontal cortex neurons.

Human prefrontal cortex (PFC) is associated with broad individual variabilities in functions linked to personality, social behaviors, and cognitive functions. The phenotype variabilities associated with brain functions can be caused by genetic or epigenetic factors. The interactions between these factors in human subjects is, as of yet, poorly understood. The heterogeneity of cerebral tissue, consisting of neuronal and nonneuronal cells, complicates the comparative analysis of gene activities in brain specimens. To approach the underlying neurogenomic determinants, we performed a deep analysis of open chromatin-associated histone methylation in PFC neurons sorted from multiple human individuals in conjunction with whole-genome and transcriptome sequencing. Integrative analyses produced novel unannotated neuronal genes and revealed individual-specific chromatin "blueprints" of neurons that, in part, relate to genetic background. Surprisingly, we observed gender-dependent epigenetic signals, implying that gender may contribute to the chromatin variabilities in neurons. Finally, we found epigenetic, allele-specific activation of the testis-specific gene nucleoporin 210 like (NUP210L) in brain in some individuals, which we link to a genetic variant occurring in <3% of the human population. Recently, the NUP210L locus has been associated with intelligence and mathematics ability. Our findings highlight the significance of epigenetic-genetic footprinting for exploring neurologic function in a subject-specific manner.-Gusev, F. E., Reshetov, D. A., Mitchell, A. C., Andreeva, T. V., Dincer, A., Grigorenko, A. P., Fedonin, G., Halene, T., Aliseychik, M., Goltsov, A. Y., Solovyev, V., Brizgalov, L., Filippova, E., Weng, Z., Akbarian, S., Rogaev, E. I. Epigenetic-genetic chromatin footprinting identifies novel and subject-specific genes active in prefrontal cortex neurons.

Neuronal Kmt2a/Mll1 histone methyltransferase is essential for prefrontal synaptic plasticity and working memory.

Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion.

Conserved chromosome 2q31 conformations are associated with transcriptional regulation of GAD1 GABA synthesis enzyme and altered in prefrontal cortex of subjects with schizophrenia.

Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS(-50kbLoop) was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS(-50kbLoop) was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS(-55kbLoop), the murine homolog to GAD1-TSS(-50kbLoop), is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS(-55kbLoop) and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.

Chromatin profiling of cortical neurons identifies individual epigenetic signatures in schizophrenia.

Both heritability and environment contribute to risk for schizophrenia. However, the molecular mechanisms of interactions between genetic and non-genetic factors remain unclear. Epigenetic regulation of neuronal genome may be a presumable mechanism in pathogenesis of schizophrenia. Here, we performed analysis of open chromatin landscape of gene promoters in prefrontal cortical (PFC) neurons from schizophrenic patients. We cataloged cell-type-based epigenetic signals of transcriptional start sites (TSS) marked by histone H3-K4 trimethylation (H3K4me3) across the genome in PFC from multiple schizophrenia subjects and age-matched control individuals. One of the top-ranked chromatin alterations was found in the major histocompatibility (MHC) locus on chromosome 6 highlighting the overlap between genetic and epigenetic risk factors in schizophrenia. The chromosome conformation capture (3C) analysis in human brain cells revealed the architecture of multipoint chromatin interactions between the schizophrenia-associated genetic and epigenetic polymorphic sites and distantly located HLA-DRB5 and BTNL2 genes. In addition, schizophrenia-specific chromatin modifications in neurons were particularly prominent for non-coding RNA genes, including an uncharacterized LINC01115 gene and recently identified BNRNA_052780. Notably, protein-coding genes with altered epigenetic state in schizophrenia are enriched for oxidative stress and cell motility pathways. Our results imply the rare individual epigenetic alterations in brain neurons are involved in the pathogenesis of schizophrenia.

In vivo epigenetic editing of Sema6a promoter reverses transcallosal dysconnectivity caused by C11orf46/Arl14ep risk gene.

Many neuropsychiatric risk genes contribute to epigenetic regulation but little is known about specific chromatin-associated mechanisms governing the formation of neuronal connectivity. Here we show that transcallosal connectivity is critically dependent on C11orf46, a nuclear protein encoded in the chromosome 11p13 WAGR risk locus. C11orf46 haploinsufficiency was associated with hypoplasia of the corpus callosum. C11orf46 knockdown disrupted transcallosal projections and was rescued by wild type C11orf46 but not the C11orf46R236H mutant associated with intellectual disability. Multiple genes encoding key regulators of axonal development, including Sema6a, were hyperexpressed in C11orf46-knockdown neurons. RNA-guided epigenetic editing of Sema6a gene promoters via a dCas9-SunTag system with C11orf46 binding normalized SEMA6A expression and rescued transcallosal dysconnectivity via repressive chromatin remodeling by the SETDB1 repressor complex. Our study demonstrates that interhemispheric communication is sensitive to locus-specific remodeling of neuronal chromatin, revealing the therapeutic potential for shaping the brain's connectome via gene-targeted designer activators and repressor proteins.

Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue.

BACKGROUND: The nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed. METHODS: Protocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries. RESULTS: We present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms. CONCLUSIONS: Our practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.

NeuN+ neuronal nuclei in non-human primate prefrontal cortex and subcortical white matter after clozapine exposure.

Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved. We exposed 26 young adult macaque monkeys for 6 months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts. After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups. Chronic clozapine exposure increases the proportion of NeuN+ nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted.

DNA Methylation Signatures of Early Childhood Malnutrition Associated With Impairments in Attention and Cognition.

BACKGROUND: Early childhood malnutrition affects 113 million children worldwide, impacting health and increasing vulnerability for cognitive and behavioral disorders later in life. Molecular signatures after childhood malnutrition, including the potential for intergenerational transmission, remain unexplored. METHODS: We surveyed blood DNA methylomes (~483,000 individual CpG sites) in 168 subjects across two generations, including 50 generation 1 individuals hospitalized during the first year of life for moderate to severe protein-energy malnutrition, then followed up to 48 years in the Barbados Nutrition Study. Attention deficits and cognitive performance were evaluated with the Connors Adult Attention Rating Scale and Wechsler Abbreviated Scale of Intelligence. Expression of nutrition-sensitive genes was explored by quantitative reverse transcriptase polymerase chain reaction in rat prefrontal cortex. RESULTS: We identified 134 nutrition-sensitive, differentially methylated genomic regions, with most (87%) specific for generation 1. Multiple neuropsychiatric risk genes, including COMT, IFNG, MIR200B, SYNGAP1, and VIPR2 showed associations of specific methyl-CpGs with attention and IQ. IFNG expression was decreased in prefrontal cortex of rats showing attention deficits after developmental malnutrition. CONCLUSIONS: Early childhood malnutrition entails long-lasting epigenetic signatures associated with liability for attention and cognition, and limited potential for intergenerational transmission.

Epigenetics and sex differences in the brain: A genome-wide comparison of histone-3 lysine-4 trimethylation (H3K4me3) in male and female mice.

Many neurological and psychiatric disorders exhibit gender disparities, and sex differences in the brain likely explain some of these effects. Recent work in rodents points to a role for epigenetics in the development or maintenance of neural sex differences, although genome-wide studies have so far been lacking. Here we review the existing literature on epigenetics and brain sexual differentiation and present preliminary analyses on the genome-wide distribution of histone-3 lysine-4 trimethylation in a sexually dimorphic brain region in male and female mice. H3K4me3 is a histone mark primarily organized as 'peaks' surrounding the transcription start site of active genes. We microdissected the bed nucleus of the stria terminalis and preoptic area (BNST/POA) in adult male and female mice and used ChIP-Seq to compare the distribution of H3K4me3 throughout the genome. We found 248 genes and loci with a significant sex difference in H3K4me3. Of these, the majority (71%) had larger H3K4me3 peaks in females. Comparisons with existing databases indicate that genes and loci with increased H3K4me3 in females are associated with synaptic function and with expression atlases from related brain areas. Based on RT-PCR, only a minority of genes with a sex difference in H3K4me3 has detectable sex differences in expression at baseline conditions. Together with previous findings, our data suggest that there may be sex biases in the use of epigenetic marks. Such biases could underlie sex differences in vulnerabilities to drugs or diseases that disrupt specific epigenetic processes.

Conserved higher-order chromatin regulates NMDA receptor gene expression and cognition.

Three-dimensional chromosomal conformations regulate transcription by moving enhancers and regulatory elements into spatial proximity with target genes. Here we describe activity-regulated long-range loopings bypassing up to 0.5 Mb of linear genome to modulate NMDA glutamate receptor GRIN2B expression in human and mouse prefrontal cortex. Distal intronic and 3' intergenic loop formations competed with repressor elements to access promoter-proximal sequences, and facilitated expression via a "cargo" of AP-1 and NRF-1 transcription factors and TALE-based transcriptional activators. Neuronal deletion or overexpression of Kmt2a/Mll1 H3K4- and Kmt1e/Setdb1 H3K9-methyltransferase was associated with higher-order chromatin changes at distal regulatory Grin2b sequences and impairments in working memory. Genetic polymorphisms and isogenic deletions of loop-bound sequences conferred liability for cognitive performance and decreased GRIN2B expression. Dynamic regulation of chromosomal conformations emerges as a novel layer for transcriptional mechanisms impacting neuronal signaling and cognition.

Maternal immune activation alters behavior in adult offspring, with subtle changes in the cortical transcriptome and epigenome.

Maternal immune activation during prenatal development, including treatment with the viral RNA mimic, polyriboinosinic-polyribocytidilic acid (poly IC), serves as a widely used animal model to induce behavioral deficits reminiscent of schizophrenia and related disease. Here, we report that massive cytokine activation after a single dose of poly IC in the prenatal period is associated with lasting working memory deficits in adult offspring. To explore whether dysregulated gene expression in cerebral cortex, contributes to cognitive dysfunction, we profiled the cortical transcriptome, and in addition, mapped the genome-wide distribution of trimethylated histone H3-lysine 4 (H3K4me3), an epigenetic mark sharply regulated at the 5' end of transcriptional units. However, deep sequencing-based H3K4me3 mapping and mRNA profiling by microarray did not reveal significant alterations in mature cerebral cortex after poly IC exposure at embryonic days E17.5 or E12.5. At a small set of genes (including suppressor of cytokine signaling Socs3), H3K4me3 was sensitive to activation of cytokine signaling in primary cultures from fetal forebrain but adult cortex of saline- and poly IC-exposed mice did not show significant differences. A limited set of transcription start sites (TSS), including Disrupted-in-Schizophrenia 1 (Disc1), a schizophrenia risk gene often implicated in gene-environment interaction models, showed altered H3K4me3 after prenatal poly IC but none of these differences survived after correcting for multiple comparisons. We conclude that prenatal poly IC is associated with cognitive deficits later in life, but without robust alterations in epigenetic regulation of gene expression in the cerebral cortex.

On detecting selective sweeps using single genomes.

Identifying the genetic basis of human adaptation has remained a central focal point of modern population genetics. One major area of interest has been the use of polymorphism data to detect so-called "footprints" of selective sweeps - patterns produced as a beneficial mutation arises and rapidly fixes in the population. Based on numerous simulation studies and power analyses, the necessary sample size for achieving appreciable power has been shown to vary from a few individuals to a few dozen, depending on the test statistic. And yet, the sequencing of multiple copies of a single region, or of multiple genomes as is now often the case, incurs considerable cost. Enard et al. (2010) have recently proposed a method to identify patterns of selective sweeps using a single genome - and apply this approach to human and non-human primates (chimpanzee, orangutan, and macaque). They employ essentially a modification of the Hudson, Kreitman, and Aguade test - using heterozygous single nucleotide polymorphisms from single individuals, and divergence data from two closely related species (human-chimpanzee, human-orangutan, and human-macaque). Given the potential importance of this finding, we here investigate the properties of this statistic. We demonstrate through simulation that this approach is neither robust to demography nor background selection; nor is it robust to variable recombination rates.