Light fragments production and isospin dependences in Sn+Ni and Sn+Al central collisions at 25MeV/A and 35MeV/A from reverse/isospin experiments

This paper presents the physical analysis results for the following reactions: 124Sn+64Ni, 124Sn+27Al, 124Sn+58Ni at 35MeV/A and 25MeV/A. The main goal of this studies was to find observables sensitive to isospin effects and to present the similarities/differences between the systems characterised by various charge asymmetry factor, defined as I = (NZ)=A. Theoretical simulations based on the Quantum Molecular Dynamics (QMD) model have been performed in order to compare them with the results of the analysis of experimental data. The first phase of the reaction was carried out with the code CHIMERA [1]. The sequential decay of hot fragments was simulated by the code COOLER [2]. The conclusions are as follows: there are observables sensitive to the isospin of the system, such as the Light Charged Particles (LCP) emission and their sensitivity is demonstrated more prominently in the analysis of central collisions at 35MeV/A. The theoretical calculations do not reproduce these relations well

Global characteristics of (197)Au+ (197)Au collisions at 23 AMeV

We present the current status of nuclear dynamics studies performed by the BREAKUP group with the 4 pi CHIMERA array, for the system Au-197 + Au-197 at 23 AMeV

SITE-SPECIFIC LABELING OF A PROTEIN LYSINE RESIDUE BY NOVEL KINETIC LABELING COMBINATORIAL LIBRARIES

The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks

Epigenetics of Epileptogenesis-Evoked Upregulation of Matrix Metalloproteinase-9 in Hippocampus

<div><p>Enhanced levels of Matrix Metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of epilepsy in humans and rodents. Lack of Mmp-9 impoverishes, whereas excess of Mmp-9 facilitates epileptogenesis. Epigenetic mechanisms driving the epileptogenesis-related upregulation of MMP-9 expression are virtually unknown. The aim of this study was to reveal these mechanisms. We analyzed hippocampi extracted from adult and pediatric patients with temporal lobe epilepsy as well as from partially and fully pentylenetetrazole kindled rats. We used a unique approach to the analysis of the kindling model results (inclusion in the analysis of rats being during kindling, and not only a group of fully kindled animals), which allowed us to separate the molecular effects exerted by the epileptogenesis from those related to epilepsy and epileptic activity. Consequently, it allowed for a disclosure of molecular mechanisms underlying causes, and not consequences, of epilepsy. Our data show that the epileptogenesis-evoked upregulation of Mmp-9 expression is regulated by removal from Mmp-9 gene proximal promoter of the two, interweaved potent silencing mechanisms–DNA methylation and Polycomb Repressive Complex 2 (PRC2)-related repression. Demethylation depends on a gradual dissociation of the DNA methyltransferases, Dnmt3a and Dnmt3b, and on progressive association of the DNA demethylation promoting protein Gadd45β to Mmp-9 proximal gene promoter <i>in vivo</i>. The PRC2-related mechanism relies on dissociation of the repressive transcription factor YY1 and the dissipation of the PRC2-evoked trimethylation on Lys27 of the histone H3 from the proximal <i>Mmp-9</i> promoter chromatin <i>in vivo</i>. Moreover, we show that the DNA hydroxymethylation, a new epigenetic DNA modification, which is localized predominantly in the gene promoters and is particularly abundant in the brain, is not involved in a regulation of MMP-9 expression during the epileptogenesis in the rat hippocampus as well as in the hippocampi of pediatric and adult epileptic patients. Additionally, we have also found that despite of its transient nature, the histone modification H3S10ph is strongly and gradually accumulated during epileptogenesis in the cell nuclei and in the proximal Mmp-9 gene promoter in the hippocampus, which suggests that H3S10ph can be involved in DNA demethylation in mammals, and not only in <i>Neurospora</i>. The study identifies <i>MMP-9</i> as the first protein coding gene which expression is regulated by DNA methylation in human epilepsy. We present a detailed epigenetic model of the epileptogenesis-evoked upregulation of <i>MMP-9</i> expression in the hippocampus. To our knowledge, it is the most complex and most detailed mechanism of epigenetic regulation of gene expression ever revealed for a particular gene in epileptogenesis. Our results also suggest for the first time that dysregulation of DNA methylation found in epilepsy is a cause rather than a consequence of this condition.</p></div

MMP-9 mRNA upregulation is accompanied by robust <i>MMP-9</i> promoter demethylation in hippocampi of epileptic patients.

<p><b>(A) <i>MMP-9 mRNA expression is substantially increased in the hippocampi of adult and pediatric epileptic patients</i></b>. For each RT-qPCR analysis, equal amounts of RNA samples isolated from control, epileptic adult and pediatric patients’ hippocampi were used. Data is presented as fold change in mRNA expression. Values are means ± SEM (*, <i>p</i> < 0.05; ***, <i>p</i> < 0.001; <i>n</i> = 6 for adults, <i>n</i> = 3–4 for pediatric patients). (<b>B) <i>MMP-9 proximal promoter methylation is strongly diminished in vivo in the hippocampi of epileptic patients</i></b>. <i>MMP-9</i> proximal promoter methylation level was evaluated by qPCR using DNA samples obtained by the immunoprecipitation of methylated DNA (MeDIP) coming from hippocampi of control as well as adult and pediatric epileptic patients. Data is presented as fold change in the methylation status. Values are means ± SEM (*, <i>p</i> < 0.05; ***, <i>p</i> < 0.001; <i>n</i> = 6 for adults, <i>n</i> = 3–4 for pediatric patients). <b>(C) <i>MMP-9 proximal promoter is hydroxymethylated at similar level in vivo in epileptic and control patients</i></b>. Level of <i>MMP-9</i> proximal promoter hydroxymethylation was evaluated using qPCR with DNA samples obtained from immunoprecipitation of hydroxymethylated DNA (hMeDIP) coming from hippocampi of control and epileptic patients. Data is presented as fold change in hydroxymethylation status. Values are means ± SEM (<i>n</i> = 6).</p

<i>Mmp-9</i> proximal promoter is progressively demethylated during PTZ-induced epileptogenesis in the rat hippocampus.

<p><b>(A) <i>Schematic presentation of our experimental approach to a detailed evaluation of the rat Mmp-9 proximal promoter methylation</i>.</b><i>In vivo</i> methylation status of ~1000 bp fragment of the <i>Mmp-9</i> proximal promoter was analyzed using the methylated DNA immunoprecipitation method (MeDIP). Methylation of CpG sites localized in the -1263/-685 and the -269/-1 bp fragments of the proximal <i>Mmp-9</i> promoter (sites 1–5 and 9–18 in the scheme) were analyzed <i>in vitro</i> by bisulfite sequencing. The methylation status of CpG sites situated in the -684/-270 bp fragment of the proximal <i>Mmp-9</i> promoter (sites 6–8 at the scheme) were evaluated <i>in vitro</i> using methylation-specific PCR (MSP). Additionally, to provide a control for the consistency of bisulfite sequencing results and data obtained by MSP, the -98/-97bp CpG site of the <i>Mmp-9</i> promoter was evaluated using both these procedures (compare middle panel in Fig 3C and 3D). (<b>B) <i>Mmp-9 proximal promoter is demethylated during epileptogenesis in vivo</i>.</b> Methylation level was revealed by MeDIP and presented as % of input. (<b>C) <i>Mmp-9 proximal promoter is progressively demethylated in most of its CpG sites during epileptogenesis in vitro</i>.</b> Methylation of CpG sites localized in -1263/-685 and -269/-1 bp fragments of <i>Mmp-9</i> promoter was evaluated using bisulfite sequencing. Graph data is presented as % of the total number of methylated and unmethylated CpG sites found in all examined clones in these regions (left panel) or as % of the total number of methylated and unmethylated CpG sites detected in the particular CpG site in the promoter fragments in all analyzed clones (central panel). Right panel presents results of DNA methylation at CpG sites located in the -684/-270 bp fragment of <i>Mmp-9</i> promoter using MSP. Data is presented as % of unmethylated DNA. (<b>D) <i>The -98/-97bp CpG site of Mmp-9 promoter is gradually demethylated during epileptogenesis in vitro</i>.</b> Methylation level was analyzed by MSP (presented as % of unmethylated DNA). (<b>E) <i>DNA hydroxymethylation of rat hippocampal Mmp-9 proximal promoter remains unchanged during epileptogenesis in vivo</i>.</b> Hydroxymethylation was assessed by hMeDIP and data is presented as % of input. In Fig 3B, 3C (right panel), 3D and 3E values are means ±SEM and <i>n</i> = 4. In Fig 3B and 3C (right panel) **, <i>p</i><0.01; ***, <i>p</i><0.001. In Fig 3D *, <i>p</i><0.05; ***, <i>p</i><0.001.</p