Preconditioning with Cortical Spreading Depression induces a sort of tolerance to a subsequent episode of ischemia. The mechanism of this tolerance is not clear. We studied if such treatment induces epigenetic chromatin modifications on the hemispheres of rats preconditioned by Cortical Spreading Depression. The contralateral hemispheres were used as control. We determined the level of H3K4 and H3K9 methylation and the mRNA amounts for the two well known H3K4 methyltransferase (MLL and SET7) in rats 24 hours after the Cortical Spreading Depression induction. Western blotting experiments have been performed using three different types of primary antibodies against mono-, di- and tri-methyl H3K4 and primary antibody anti-dimethyl H3K9. In the same samples we checked if the H3 histones were replaced by the H3.3 histone variants that could be an additional marker of chromatin modifications. The level of mono- and di-methyl H3K4 was significantly lower in samples of the treated hemispheres than those of the contralateral hemispheres (40% and about 60%, respectively) while the level of tri-methylation remained unchanged. The level of di-methyl H3K9 was almost 60% higher in the treated hemispheres than the contralateral hemispheres. The treatment for Cortical Spreading Depression affected also the level of expression of H3K4 histone methyltransferase MLL and SET7 that decreased in the treated hemispheres in comparison to the control hemispheres (80% and 40%, respectively). The treatment for Cortical Spreading Depression instead had no effects on the overall amounts of mRNA for H3 and H3.3 histones. In conclusion epigenetic chromatin modifications are evident in rats 24 hours after the Cortical Spreading Depression induction.
20211609

Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.
20211173

Abstract Background. DNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine -NH(3)(+) groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci. Methods. We analysed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin(+)) and progenitor lineage marker-negative (Lin(-)) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence. Results. We observed that Lin(-) and Lin(+) cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4(-/-) mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4. Conclusions. Overall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.
20210674

One of the most fundamental questions in control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling metazoan gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on recent developments in characterizing the functional links between methylation status of the DNA and of two particularly important histone marks. Mammalian DNA methylation is intricately connected to the presence of unmodified lysine 4 and methylated lysine 9 residues in histone H3. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. The structural and functional interactions among members of this network are critical to processes that include imprinting and differentiation, and dysregulation of which is associated with disorders ranging from inflammation to cancer.
20210320

Chromatin immunoprecipitation in combination with DNA-microarray hybridization (ChIP-chip) allows the identification of chromatin regions that are associated with modified forms of histones on a genomic scale. The ChIP-chip workflow consists of the following steps: generation of biological material, in vivo formaldehyde-fixation of protein-DNA and protein-protein interactions, chromatin preparation and shearing, immunoprecipitation of chromatin with specific antibodies, fixation reversal and DNA purification, DNA amplification, microarray hybridization, and data analysis. In Part A of this chapter, we describe molecular methods of the experimental procedure employed to identify chromosomal regions of Arabidopsis thaliana associated with H3K27me3. In addition, some general information on the microarray platform from Roche-NimbleGen will be provided. Part B of this chapter focuses on ChIP-chip data analysis of H3K27me3 on the Roche-NimbleGen platform.
20204874

Eukaryotic genomic DNA is orderly compacted to fit into the nucleus and to inhibit accessibility of specific sequences. DNA is manipulated in many different ways by bound RNA and proteins within the composite material known as chromatin. All of the biological processes that require access to genomic DNA (such as replication, recombination and transcription) therefore are dependent on the precise characteristics of chromatin in eukaryotes. This distinction underlies a fundamental property of eukaryotic versus prokaryotic gene regulation such that chromatin structure must be regulated to precisely repress or relieve repression of particular regions of the genome in an appropriate spatio-temporal manner. As well as playing a key role in structuring genomic DNA, histones are subject to site-specific modifications that can influence the organization of chromatin structure. This review examines the molecular processes regulating site-specific histone acetylation, methylation and phosphorylation with an emphasis on how these processes underpin differentiation-regulated transcription.
20204433

Extracellular factors control the angiogenic switch in endothelial cells (EC) via competing survival and apoptotic pathways. Previously, we showed that pro- and anti-angiogenic factors target the same signaling molecules, which thereby become pivots of angiogenic balance. Here we show that in remodeling endothelium (EC and EC precursors) natural angiogenic inhibitors enhance NFkappaB DNA binding that is critical for anti-angiogenesis; blocking the NFkappaB pathway abolished multiple anti-angiogenic events in vitro and in vivo. NFkappaB induction by anti-angiogenic molecules has a dual effect on transcription. NFkappaB acts as an activator of pro-apoptotic FasL and as a repressor of pro-survival cFLIP. On the FasL promoter, NFkappaB increases the recruitment of HAT p300, and acetylated histones H3 and H4. Conversely, on cFLIP promoter, NFkappaB increases HDAC1, decreases p300 and histone acetylation, and reduces the recruitment of NFAT, a transcription factor critical for cFLIP expression. Finally, we found a biphasic effect, when HDAC inhibitors (HDACi) were used to test the dependence of PEDF activity on histone acetylation. The cooperative effect seen at low doses switches to antagonistic as the concentrations increase. Our study defines an interactive transcriptional network underlying angiogenic balance and points to HDACi as tools to manipulate the angiogenic switch.
20203265

Transcriptional states are formed and maintained by the interaction and post-translational modification (PTM) of several chromatin proteins, such as histones and high mobility group (HMG) proteins. Among these, HMGA1a, a small heterochromatin-associated nuclear protein has been shown to be post-translationally modified, and some of these PTMs have been linked to apoptosis and cancer. In cancerous cells, HMGA1a PTMs differ between metastatic and nonmetastatic cells, suggesting the existence of an HMGA1a PTM code analogous to the "histone code." In this study, we expand on current knowledge by comprehensively characterizing PTMs on HMGA1a purified from human cells using both nanoflow liquid chromatography collision activated dissociation mediated Bottom Up and electron-transfer dissociation facilitated middle and Top Down mass spectrometry (MS). We find HMGA1a to be pervasively modified with many types of modifications such as methylation, acetylation, and phosphorylation, including finding novel sites. While Bottom Up MS identified lower level modification sites, Top and Middle Down MS were utilized to identify the most commonly occurring combinatorially modified forms. Remarkably, although we identify several individual modification sites through our Bottom Up and Middle Down MS analyses, we find relatively few combinatorially modified forms dominate the population through Top Down proteomics. The main combinatorial PTMs we find through the Top Down approach are N-terminal acetylation, Arg25 methylation along with phosphorylation of the three most C-terminal serine residues in primarily a diphosphorylated form. This report presents one of the most detailed analyses of HMGA1a to date and illustrates the strength of using a combined MS effort.
20202861

The rich diversity within each of the five histone families (H1, H2A, H2B, H3, H4) can hardly be reconciled with the notion of homogenizing evolution. The prevalence of birth-and-death long-term evolution over concerted evolution has already been demonstrated in the linker histone H1 family as well as for the H2A, H3 and H4 core histone families. However, information about histone H2B is lacking. In the present work we have analyzed the diversity of the members of this histone family across different eukaryotic genomes and have characterized the mechanisms involved in their long-term evolution. Our results reveal that, quite in contrast with other histones, H2B variants are subject to a very rapid process of diversification which primarily affects the male germinal cell lineage and involves their functional specialization probably as a consequence of neofunctionalization and subfunctionalization events after gene duplication. The overall parallelism observed between the molecular phylogenies and the relationships among the electrostatic potentials of the different variants suggests that the latter may have played a major structural selective constraint during H2B evolution. It thus seems that the reorganization of chromatin structure during spermiogenesis might have affected the evolutionary constraints driving histone H2B evolution, leading to an increase in diversity.
20194426

Proper cell cycle-dependent expression of replication-dependent histones is essential for packaging of DNA into chromatin during replication. We previously showed that cyclin-dependent kinase-9 (CDK9) controls histone H2B monoubiquitination (H2Bub1) to direct the recruitment of specific mRNA 3' end processing proteins to replication-dependent histone genes and promote proper pre-mRNA 3' end processing. We now show that p53 decreases the expression of the histone-specific transcriptional regulator Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) by inducing a G1 cell-cycle arrest, thereby affecting E2F-dependent transcription of the NPAT gene. Furthermore, NPAT is essential for histone mRNA 3' end processing and recruits CDK9 to replication-dependent histone genes. Reduced NPAT expression following p53 activation or small interfering RNA knockdown decreases CDK9 recruitment and replication-dependent histone gene transcription but increases the polyadenylation of remaining histone mRNAs. Thus, we present evidence that the induction of a G1 cell-cycle arrest (for example, following p53 accumulation) alters histone mRNA 3' end processing and uncover the first mechanism of a regulated switch in the mode of pre-mRNA 3' end processing during a normal cellular process, which may be altered during tumorigenesis.Oncogene advance online publication, 1 March 2010; doi:10.1038/onc.2010.42.
20190802

OBJECTIVE: PR (PRDI-BF1 and RIZ) domain proteins (PRDM) are a subfamily of the kruppel-like zinc finger gene products and play key roles during cell differentiation and malignant transformation. PRDM5 (PR domain containing 5 PFM2) is a new PR-domain-containing gene. The purpose of the present study was to examine the expression of PRDM5 and evaluate its carcinogenesis in cervical cancer. The relationship between DNA methylation and transcriptional silencing of PRDM5 was investigated in cervical cancer. METHODS: PRDM5 expression was examined in cervical cancer cell lines and cervical tissues (12 normal and 42 cancerous) by using RT polymerase chain reaction (PCR). Methylation status of the PRDM5 promoter was studied using methylation-specific PCR (MSP). RESULTS: PRDM5 expression is reduced or lost in cervical cancers, compared with normal cervical tissues (P < 0.05). The current study results also showed that loss of PRDM5 is mediated by aberrant cytosine methylation of the PRDM5 promoter. There were 40.5% of carcinomas methylated, while none of normal tissues were methylated. PRDM5 mRNA expression was significantly higher (P = 0.000) in unmethylated (0.2634 +/- 0.0674, mean +/- SD), compared with methylated tissues (0.1007 +/- 0.0993, mean +/- SD). Last, treatment with a DNA methyltransferase inhibitor led to reactivation of PRDM5 expression in cell lines that had negligible PRDM5 expression at baseline. CONCLUSIONS: Reduced expression of PRDM5 may play an important role in the pathogenesis and/or development of cervical cancer, and is considered to be caused in part by aberrant DNA methylation.
20213097

DNA methylation in gene promoters causes gene silencing and is a common event in cancer development and progression. The ability of aberrant methylation events to serve as diagnostic and prognostic markers is being appreciated for many cancers, including prostate cancer. Using quantitative MethyLight technology, we evaluated the relationship between HOXD3 methylation and clinicopathological parameters including biochemical recurrence, pathological stage, Gleason score (GS), and Gleason pattern in a series of 232 radical prostatectomies performed between 1998 and 2001. HOXD3 methylation was significantly greater in GS 7 cancers vs GS 20212450

The DNA of most vertebrates is depleted in CpG dinucleotide: a C followed by a G in the 5' to 3' direction. CpGs are the target for DNA methylation, a chemical modification of cytosine (C) heritable during cell division and the most well-characterized epigenetic mechanism. The remaining CpGs tend to cluster in regions referred to as CpG islands (CGI). Knowing CGI locations is important because they mark functionally relevant epigenetic loci in development and disease. For various mammals, including human, a readily available and widely used list of CGI is available from the UCSC Genome Browser. This list was derived using algorithms that search for regions satisfying a definition of CGI proposed by Gardiner-Garden and Frommer more than 20 years ago. Recent findings, enabled by advances in technology that permit direct measurement of epigenetic endpoints at a whole-genome scale, motivate the need to adapt the current CGI definition. In this paper, we propose a procedure, guided by hidden Markov models, that permits an extensible approach to detecting CGI. The main advantage of our approach over others is that it summarizes the evidence for CGI status as probability scores. This provides flexibility in the definition of a CGI and facilitates the creation of CGI lists for other species. The utility of this approach is demonstrated by generating the first CGI lists for invertebrates, and the fact that we can create CGI lists that substantially increases overlap with recently discovered epigenetic marks. A CGI list and the probability scores, as a function of genome location, for each species are available at http://www.rafalab.org.
20212320

The involvement of epigenetic alterations in mediating effects of estrogens on memory is unknown. The present study determined whether histone acetylation and DNA methylation are critical for the potent estrogen 17beta-estradiol (E(2)) to enhance object recognition memory. We show that dorsal hippocampal E(2) infusion increases acetylation of dorsal hippocampal histone H3, but not H4-an effect blocked by dorsal hippocampal inhibition of ERK activation. Further, intrahippocampal inhibition of ERK activation or DNA methyltransferase (DNMT) activity blocked the memory-enhancing effects of E(2). Consistent with these effects, E(2) decreased levels of HDAC2 protein and increased DNMT expression in the dorsal hippocampus. These findings provide evidence that the beneficial effects of E(2) on memory consolidation are associated with epigenetic alterations, and suggest these can be triggered by dorsal hippocampal ERK signaling.
20212170

Cytochrome aromatase p450, encoded by the gene CYP19, catalyzes the synthesis of estrogens from androgens. In post-menopausal women, adipose becomes the major site for estrogen production, where basal CYP19 transcription is driven by distal promoter I.4. In breast adipose fibroblasts (BAFs), CYP19 expression is elevated in the presence of tumor-derived factors through use of promoters I.3 and II. We show for the first time that DNA methylation contributes to CYP19 regulation in BAFs and breast cell lines. Promoter I.4 and I.3/II-derived mRNA were not dependent on the CpG methylation status within respective promoters. However, inhibition of DNA methylation with 5-aza-2'-deoxycytidine resulted in a significant approximately 40-fold induction in CYP19 mRNA expression in BAFs and breast cell lines. These studies uncover a new layer of complexity in the regulation of aromatase where CYP19 appears to be inhibited by DNA methylation and evokes the possibility that disruption to this epigenetic regulation may give rise to an increase in aromatase levels in the breast.
20211687

OBJECTIVE: DNA promoter methylation is an epigenetic phenomenon for long-term gene silencing during tumorigenesis. The purpose of this study is to identify novel hypermethylated loci associated with clinicopathologic variables in endometrioid endometrial carcinomas. METHODS: To find hypermethylated promoter loci, we used differential methylation hybridization coupling with microarray and further validated by combined bisulfite restriction analysis and MassARRAY assay. Methylation levels of candidate loci were corrected with clinicopathologic factors of endometrial carcinomas. RESULTS: Increased promoter methylation of CIDE, HAAO and RXFP3 was detected in endometrial carcinomas compared with adjacent normal tissues, and was associated with decreased gene expression of all three genes. In a clinical cohort, promoter hypermethylation on CIDEA, HAAO and RXFP3 was detected in 85, 63 and 71% of endometrial carcinomas, respectively (n=118, P<0.001) compared with uninvolved normal endometrium. Methylation status of CIDEA, HAAO and RXFP3 had significant association with microsatellite instability in tumors (P<0.001). Furthermore, methylation levels of HAAO were further found to relate to disease-free survivals (P=0.034). CONCLUSIONS: Hypermethylation of CIDEA, HAAO and RXFP3 promoter regions appears to be a frequent event in endometrial carcinomas. Hypermethylation at these loci is strongly associated with microsatellite instability status. Moreover, HAAO methylation predicts disease-free survival in this cohort of patients with endometrioid endometrial cancer.
20211485

Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long term plasticity triggered by pharmacological agents.
20211261

One of the most fundamental questions in control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling metazoan gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on recent developments in characterizing the functional links between methylation status of the DNA and of two particularly important histone marks. Mammalian DNA methylation is intricately connected to the presence of unmodified lysine 4 and methylated lysine 9 residues in histone H3. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. The structural and functional interactions among members of this network are critical to processes that include imprinting and differentiation, and dysregulation of which is associated with disorders ranging from inflammation to cancer.
20210320

AIMS AND BACKGROUND: Exfoliated cells in human stool offer excellent opportunities to non-invasively detect molecular markers associated with colorectal tumorigenesis, and to evaluate the effects of exposures to exogenous and endogenous carcinogenic or chemopreventive substances. This pilot study investigated the feasibility of determining DNA methylation and RNA expression simultaneously in stool specimens treated with a single type of nucleic acid preservatives. METHODS: Stool specimens from 56 volunteers that were preserved up to a week with RNA later were used in this study. Bisulfite sequencing was used to determine methylation at 27 CpG loci on the estrogen receptor 1 (ESR1) promoter. Taqman assay was used for quantitative reverse transcription polymerase chain reactions to measure cyclooxygenase 2 (COX2) and epidermal growth factor receptor (EGFR) mRNA expression. Subjects' basic demographic and other selected risk factors for colorectal cancer were captured through questionnaires and correlated with the levels of these markers. RESULTS: Less than 10% of the samples failed in individual assays. Overall, 24.0% of the CpG loci on the ESR1 promoter were methylated. COX2 expression and alcohol use were positively correlated; an inverse association was present between EGFR expression and cigarette smoking; and subjects using anti-diabetic medication had higher ESR1 methylation. In addition, higher EGFR expression levels were marginally associated with history of polyps and family history of colorectal cancer. CONCLUSIONS: The present study demonstrates that simultaneous analyses for DNA and RNA markers are feasible in stool samples treated with a single type of nucleotide preservatives. Among several associations observed, the association between EGFR expression and polyps deserves further investigation as a potential target for colorectal cancer screening. Larger studies are warranted to confirm some of our observations.
20210241

Epigenetic states and certain environmental responses in mammals and seed plants can persist in the next sexual generation. These transgenerational effects have potential adaptative significance as well as medical and agronomic ramifications. Recent evidence suggests that some abiotic and biotic stress responses of plants are transgenerational. For example, viral infection of tobacco plants and exposure of Arabidopsis thaliana plants to UVC and flagellin can induce transgenerational increases in homologous recombination frequency (HRF). Here we show that exposure of Arabidopsis plants to stresses, including salt, UVC, cold, heat and flood, resulted in a higher HRF, increased global genome methylation, and higher tolerance to stress in the untreated progeny. This transgenerational effect did not, however, persist in successive generations. Treatment of the progeny of stressed plants with 5-azacytidine was shown to decrease global genomic methylation and enhance stress tolerance. Dicer-like (DCL) 2 and DCL3 encode Dicer activities important for small RNA-dependent gene silencing. Stress-induced HRF and DNA methylation were impaired in dcl2 and dcl3 deficiency mutants, while in dcl2 mutants, only stress-induced stress tolerance was impaired. Our results are consistent with the hypothesis that stress-induced transgenerational responses in Arabidopsis depend on altered DNA methylation and smRNA silencing pathways.
20209086

The International Union of Pure and Applied Chemistry (IUPAC) code specified nearly 25 years ago provides a nomenclature for incompletely specified nucleic acids (Cornish-Bowden 1984). The IUPAC code has been applied in a wide-ranging manner, contributing to many biologically and chemically meaningful representations, including: 1) recognition sequences (e.g., restriction enzymes, protein and RNA binding sites, consensus signals), 2) codon degeneracy, 3) sequence base calling ambiguity 4) representation of ancestral states in phylogenetics and 5) to a vast extent in the fields of genetics and genomics in representing polymorphic nucleic acids, e.g., single nucleotide polymorphisms (SNPs). However, no system currently exists that allows for the informatics representation of the relative abundance at polymorphic nucleic acids (e.g., SNPs) in a single specified character, or a string of characters. Here I propose such an information code as a natural extension to the IUPAC nomenclature code, and present some potential uses and limitations to such a code. The original IUPAC code remains useful in all its previous applications and is also compatible as a subset of the extended code proposed here. The extended IUPAC code allows for new nucleic acid representations, in single characters or character strings, with potential applications in genetics, cross-species or cross-strain comparison, sequence alignment, bioinformatics, genome assembly, database design and querying, and chemical sequencing and synthesis. The primary anticipated use of this extended nomenclature code is to assist in the representation of the rapidly growing space of information on human genetic variation.
20202974

BACKGROUND: RNA structure prediction problem is a computationally complex task, especially with pseudo-knots. The problem is well-studied in existing literature and predominantly uses highly coupled Dynamic Programming (DP) solutions. The problem scale and complexity become embarrassingly humungous to handle as sequence size increases. This makes the case for parallelization. Parallelization can be achieved by way of networked platforms (clusters, grids, etc) as well as using modern day multi-core chips. METHODS: In this paper, we exploit the parallelism capabilities of the IBM Cell Broadband Engine to parallelize an existing Dynamic Programming (DP) algorithm for RNA secondary structure prediction. We design three different implementation strategies that exploit the inherent data, code and/or hybrid parallelism, referred to as C-Par, D-Par and H-Par, and analyze their performances. Our approach attempts to introduce parallelism in critical sections of the algorithm. We ran our experiments on SONY Play Station 3 (PS3), which is based on the IBM Cell chip. RESULTS: Our results suggest that introducing parallelism in DP algorithm allows it to easily handle longer sequences which otherwise would consume a large amount of time in single core computers. The results further demonstrate the speed-up gain achieved in exploiting the inherent parallelism in the problem and also elicits the advantages of using multi-core platforms towards designing more sophisticated methodologies for handling a fairly long sequence of RNA. CONCLUSION: The speed-up performance reported here is promising, especially when sequence length is long. To the best of our literature survey, the work reported in this paper is probably the first-of-its-kind to utilize the IBM Cell Broadband Engine (a heterogeneous multi-core chip) to implement a DP. The results also encourage using multi-core platforms towards designing more sophisticated methodologies for handling a fairly long sequence of RNA to predict its secondary structure.
20122209

The rapidly emerging science of epigenetics and epigenomic medicine promises to reveal novel insights into the susceptibility to and the onset and progression of epileptic disorders. Epigenetic regulatory mechanisms are now implicated in orchestrating aspects of neural development (e.g., cell fate specification and maturation), homeostasis and stress responses (e.g., immediate early gene transcription), and neural network function (e.g., excitation-inhibition coupling and activity-dependent plasticity). These same neurobiological processes are responsible for determining the heterogeneous features of complex epileptic disease states. Thus, we highlight recent evidence that is beginning to elucidate the specific roles played by epigenetic mechanisms, including DNA methylation, histone code modifications and chromatin remodeling, noncoding RNAs and RNA editing, in human epilepsy syndromes and in the process of epileptogenesis. The highly integrated layers of the epigenome are responsible for the cell type specific and exquisitely environmentally responsive deployment of genes and functional gene networks that underlie the molecular pathophysiology of epilepsy and its associated comorbidities, including but not limited to neurotransmitter receptors (e.g., GluR2, GLRA2, and GLRA3), growth factors (e.g., BDNF), extracellular matrix proteins (e.g., RELN), and diverse transcriptional regulators (e.g., CREB, c-fos, and c-jun). These important observations suggest that future epigenetic studies are necessary to better understand, classify, prevent, and treat epileptic disorders.
20188170

We developed orthogonal ribosome-mRNA pairs in which the orthogonal ribosome (O-ribosome) specifically translates the orthogonal mRNA and the orthogonal mRNA is not a substrate for cellular ribosomes. O-ribosomes have been used to create new cellular circuits to control gene expression in new ways, they have been used to provide new information about the ribosome, and they form a crucial part of foundational technologies for genetic code expansion and encoded and evolvable polymer synthesis in cells. The production of O-ribosomes in the cell makes it challenging to study the properties of O-ribosomes in vitro, because no method exists to purify functional O-ribosomes from cellular ribosomes and other cellular components. Here we present a method for the affinity purification of O-ribosomes, via tagging of the orthogonal 16S ribosomal RNA. We demonstrate that the purified O-ribosomes are pure by primer extension assays, and structurally homogenous by gel electrophoresis and sucrose gradients. We demonstrate the utility of this purification method by providing a preliminary in vitro characterization of Ribo-X, an O-ribosome previously evolved for enhanced unnatural amino acid incorporation in response to amber codons. Our data suggest that the basis of Ribo-X's in vivo activity is a decreased affinity for RF1.
20185573

Abstract AKI is being increasingly shown to be a risk factor for CKD, but little is known about the possible mechanistic links. We hypothesized that analysis of the genomic signature in the repair stage after AKI would reveal pathways that could link AKI and CKD. Unilateral renal pedicle clamping for 45 mins was performed in male C57BL/6J mice. Mice were sacrificed at 3, 10 or 28 days after IRI. Total RNA was isolated from kidney, and analyzed using Illumina mouse array. Among 24,600 tested genes, 242, 146 and 46 genes were up-regulated at day 3, 10 and 28 after IRI, and 85, 35 and 0 genes were down regulated, respectively. Gene ontology analysis showed that gene expression changes were primarily related to immune and inflammatory pathways both early and late after AKI. The most highly upregulated genes late after AKI were hepatitis A virus cellular receptor 1 and lipocalin 2, which code for KIM-1 and NGAL, respectively. This was unexpected since they are both primarily potential biomarkers of the early stage of AKI. Furthermore, increases observed in gene expression in Abp 1, VCAM-1 and endothelin 1 could explain the salt sensitive hypertension that can follow AKI. These data suggested that: 1) persistent inflammation and immune responses late after AKI could contribute to the pathogenesis of CKD, 2) late upregulation of KIM-1 and NGAL could be a useful marker for sustained renal injury after AKI, and 3) hypertension related gene changes could underlie mechanisms for persistent renal and vascular injury after AKI. Key words: ischemic reperfusion injury, gene expression profiling, chronic kidney disease.
20181666

The relationship between TNF and immune pathology forced an intense research into the regulation of its biosynthesis that extends to multiple mechanisms controlling the utilization of its mRNA. These posttranscriptional mechanisms gradually and variably impose a series of flexible rate-limiting controls to modify the abundance of the TNF mRNA and the rate of its translation in response to environmental signals. Mechanistically, these controls consist of signaling networks converging to RNA-binding proteins and microRNAs, which in turn target a code of secondary or tertiary ribonucleotide structures located on the TNF mRNA. The outcome of these interactions is the stringent control of this mRNA's maturation, localization, turnover and translation. A wealth of molecular and genetic data highlighted that if these posttranscriptional interactions fail, they perturb cellular responses to provide the impetus for TNF-mediated inflammatory disease. Here, we highlight the parameters guiding the posttranscriptional regulation of TNF mRNA and their relevance to homeostasis and pathology.
20173387

Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from P site to exit site is catalyzed in eukaryotes by the translocase elongation factor 2 (EF-2) and in prokaryotes by its homolog EF-G. During tRNA movement one or more "hybrid" states (A/P) is occupied, but molecular details of them and of the translocation process are limited. Here we show by cryo-electron microscopy that a population of mammalian ribosomes stalled at an mRNA pseudoknot structure contains structurally distorted tRNAs in two different A/P hybrid states. In one (A/P'), the tRNA is in contact with the translocase EF-2, which induces it. In the other (A/P''), the translocase is absent. The existence of these alternative A/P intermediate states has relevance to our understanding of the mechanics and kinetics of translocation.
20159470

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.
20154731

BACKGROUND: Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length. RESULTS: Transcriptional profiling of A. pleuropneumoniae recovered from the lung of a pig suffering from a natural infection or following growth of the bacterial isolate in BHI medium was performed. An RNA extraction protocol combining beadbeating and hot-acid-phenol was developed in order to maximize bacterial mRNA yields and quality following total RNA extraction from lung lesions. Nearly all A. pleuropneumoniae transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed in vivo during the acute phase of the infection. Our results indicate that, for example, gene apxIVA from an operon coding for RTX toxin ApxIV is highly up-regulated in vivo, and that two genes from the operon coding for type IV fimbriae (APL_0878 and APL_0879) were also up-regulated. These transcriptional profiling data, combined with previous comparative genomic hybridizations performed by our group, revealed that 66 out of the 72 up-regulated genes are conserved amongst all serotypes and that 3 of them code for products that are predicted outer membrane proteins (genes irp and APL_0959, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively) or lipoproteins (gene APL_0920). Only 4 of 72 up-regulated genes had previously been identified in controled experimental infections. CONCLUSIONS: These genes that we have identified as up-regulated in vivo, conserved across serotypes and coding for potential outer membrane proteins represent potential candidates for the development of a cross-protective vaccine against porcine pleuropneumonia.
20141640

Non-coding RNAs (ncRNAs) are RNA molecules that exclude mRNA, tRNA and rRNA, and do not code proteins. ncRNAs play a various roles in the regulation of important vital activities in many organisms such as bacteria, fungi and mammals. Recent researches have shown that ncRNAs, as oncogenes or tumor suppressor genes, have tremendous impacts on the occurrence and development of tumors. Meanwhile, ncRNAs have become a new type of tumor markers and new targets for cancer treatment. This review describes the research progresses of ncRNAs such as small interference RNA and microRNA, and their roles in carcinogenesis.
20140869