Distribution Of Binding Sites Research Articles

Insight into the Structure and Activity of Surface-Engineered Lipase Biofluids.

Despite a successful application of solvent-free liquid protein (biofluids) concept to a number of commercial enzymes, the technical advantages of enzyme biofluids as hyperthermal stable biocatalysts cannot be fully utilized as up to 90-99% of native activities are lost when enzymes were made into biofluids. With a two-step strategy (site-directed mutagenesis and synthesis of variant biofluids) on Bacillus subtilis lipase A (BsLA), we elucidated a strong dependency of structure and activity on the number and distribution of polymer surfactant binding sites on BsLA surface. Here, it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design, either with enhanced activity in aqueous solution in native form, or with improved physical property and increased activity in solvent-free system in the form of a protein liquid. This work answered some fundamental questions about the surface characteristics for construction of biofluids, useful for identifying new strategies for developing advantageous biocatalysts.

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

Regional vesicular acetylcholine transporter distribution in human brain: A [18 F]fluoroethoxybenzovesamicol positron emission tomography study.

Prior efforts to image cholinergic projections in human brain in vivo had significant technical limitations. We used the vesicular acetylcholine transporter (VAChT) ligand [18 F]fluoroethoxybenzovesamicol ([18 F]FEOBV) and positron emission tomography to determine the regional distribution of VAChT binding sites in normal human brain. We studied 29 subjects (mean age 47 [range 20-81] years; 18 men; 11 women). [18 F]FEOBV binding was highest in striatum, intermediate in the amygdala, hippocampal formation, thalamus, rostral brainstem, some cerebellar regions, and lower in other regions. Neocortical [18 F]FEOBV binding was inhomogeneous with relatively high binding in insula, BA24, BA25, BA27, BA28, BA34, BA35, pericentral cortex, and lowest in BA17-19. Thalamic [18 F]FEOBV binding was inhomogeneous with greatest binding in the lateral geniculate nuclei and relatively high binding in medial and posterior thalamus. Cerebellar cortical [18 F]FEOBV binding was high in vermis and flocculus, and lower in the lateral cortices. Brainstem [18 F]FEOBV binding was most prominent at the mesopontine junction, likely associated with the pedunculopontine-laterodorsal tegmental complex. Significant [18 F]FEOBV binding was present throughout the brainstem. Some regions, including the striatum, primary sensorimotor cortex, and anterior cingulate cortex exhibited age-related decreases in [18 F]FEOBV binding. These results are consistent with prior studies of cholinergic projections in other species and prior postmortem human studies. There is a distinctive pattern of human neocortical VChAT expression. The patterns of thalamic and cerebellar cortical cholinergic terminal distribution are likely unique to humans. Normal aging is associated with regionally specific reductions in [18 F]FEOBV binding in some cortical regions and the striatum.

Experimental Cerebral Ischemia Affects the Expression of Circular RNA Genes of Metabotropic Glutamate Receptors mGluR3 and mGluR5 in Rat Brain

Circular RNAs (circRNAs) are a new RNA type that show predominantly brain-specific expression pattern in mammals. Their individual representatives, acting as competitive endogenous RNAs, can bind microRNAs and contribute to the protection of functional transcripts. In the model of transient cerebral ischemia in rats, we studied the expression of mGluR3 and mGluR5 glutamate metabotropic receptor genes (Grm3 and Grm5). These genes are important participants in the metabolic pathways associated with neurosignaling. In the present study, we found that the rat Grm3 and Grm5 genes, in addition to mRNA, encode circRNAs, which are conserved in humans and rodents. In subcortical brain structures of rats containing a lesion focus, the level of these circRNAs is more stable than that of the corresponding mRNA. Using STarMirDB database analysis, the distribution of the microRNA binding sites along the mRNA molecules of human GRM3 and GRM5 genes which are homologous to the corresponding rat genes was elucidated. It has been revealed that sufficiently large number of binding sites is located within exons, which are also part of conservative circRNAs. The results may indicate the functional role of the circRNAs of the investigated genes as competitive endogenous RNAs in the response of brain cells to ischemia.

Conformational Plasticity in Tyrosine Kinase Inhibitor-Kinase Interactions Revealed with Fluorescence Spectroscopy and Theoretical Calculations.

To understand drug-protein dynamics, it is necessary to account for drug molecular flexibility and binding site plasticity. Herein, we exploit fluorescence from a tyrosine kinase inhibitor, AG1478, as a reporter of its conformation and binding site environment when complexed with its cognate kinase. Water-soluble kinases, aminoglycoside phosphotransferase APH(3')-Ia and mitogen-activated protein kinase 14 (MAPK14), were chosen for this study. On the basis of our prior work, the AG1478 conformation (planar or twisted) was inferred from the fluorescence excitation spectrum and the polarity of the AG1478-binding site was deduced from the fluorescence emission spectrum, while red-edge excitation shift (REES) probed the heterogeneity of the binding site (protein conformation and hydration) distributions in the protein conformational ensemble. In the AG1478-APH(3')-Ia complex, both twisted (or partially twisted) and planar AG1478 conformations were evidenced from emission wavelength-dependent excitation spectra. The binding site environment provided by APH(3')-Ia was moderately polar (λmax = 480 nm) with evidence for considerable heterogeneity (REES = 34 nm). In contrast, in the AG1478-MAPK14 complex, AG1478 was in a predominantly planar conformation with a lower degree of conformational heterogeneity. The binding site environment provided by the MAPK14 protein was of relatively low polarity (λmax = 430 nm) with a smaller degree of heterogeneity (REES = 11 nm). The results are compared with the available X-ray data and discussed in the context of our current understanding of tyrosine kinase inhibitor conformation and protein conformational ensembles.

PREP1 tumor suppressor protects the late-replicating DNA by controlling its replication timing and symmetry

The synthesis of middle-to-late-replicating DNA can be affected independently of the rest of the genome by down-regulating the tumor suppressor PREP1 (PKNOX1). Indeed, DNA combing shows that PREP1 down-regulation affects DNA replication rate, increases the number of simultaneously firing origins and the asymmetry of DNA replication, leading to DNA damage. Genome-wide analysis of replication timing by Repli-seq shows that, upon PREP1 down-regulation, 25% of the genome is replicated earlier in the S-phase. The targeted DNA sequences correspond to Lamin-Associated Domains (LADs), and include late-replicating (LRRs) and temporal transition regions (TTRs). Notably, the distribution of PREP1 DNA binding sites and of its target genes indicates that DNA replication defects are independent of the overall PREP1 transcriptional activity. Finally, PREP1 down-regulation causes a substantial decrease in Lamin B1 levels. This suggests that DNA is released from the nuclear lamina earlier than in the control cells and is available for replication, thus explaining timing defects and DNA damage.This is the first evidence that the replication timing of a specific fraction of the human genome is affected by PREP1 tumor suppressor. This previously unknown function might significantly contribute to the genomic instability observed in human tumors.

Ligand Displacement Exposes Binding Site Heterogeneity on CdSe Nanocrystal Surfaces

Nanocrystal-ligand interactions and ligand exchange processes are usually described by a uniform distribution of equal binding sites. Here, we analyze this assumption by a quantitative study of the displacement of Z-type cadmium oleate ligands from CdSe nanocrystals by addition of L-type ligands. First, we determined the stoichiometry of the displacement reaction by analyzing the equilibrium upon dilution using solution nuclear magnetic resonance spectroscopy. We found that 1 equivalent of tetramethylethylene-1,2-diamine (TMEDA) or two equivalents of n-butylamine or benzylamine bind to the displaced cadmium oleate. We only reached a comprehensive description of the displacement isotherm by including two types of binding sites with a different equilibrium constant. We corroborated this finding by density functional theory calculations on a CdSe model nanocrystal, which show that even single facets contain a broad variety of binding sites. Finally, we analyzed the thermodynamics of the displacement equilibr...

The Pattern of microRNA Binding Site Distribution.

Micro-RNA (miRNA or miR) regulates at least 60% of the genes in the human genome through their target sites at mRNA 3’-untranslated regions (UTR), and defects in miRNA expression regulation and target sites are frequently observed in cancers. We report here a systematic analysis of the distribution of miRNA target sites. Using the evolutionarily conserved miRNA binding sites in the TargetScan database (release 7.1), we constructed a miRNA co-regulation network by connecting genes sharing common miRNA target sites. The network possesses characteristics of the ubiquitous small-world network. Non-hub genes in the network—those sharing miRNA target sites with small numbers of genes—tend to form small cliques with their neighboring genes, while hub genes exhibit high levels of promiscuousness in their neighboring genes. Additionally, miRNA target site distribution is extremely uneven. Among the miRNAs, the distribution concentrates on a small number of miRNAs, in that their target sites occur in an extraordinarily large number of genes, that is, they have large numbers of target genes. The distribution across the genes follows a similar pattern; the mRNAs of a small proportion of the genes contain extraordinarily large numbers of miRNA binding sites. Quantitatively, the patterns fit into the P(K) ∝ K−α relationship (P(K): the number of miRNAs with K target genes or genes with K miRNA sites; α: a positive constant), the mathematical description of connection distribution among the nodes and a defining characteristic of the so-called scale-free networks—a subset of small-world networks. Notably, well-known tumor-suppressive miRNAs (Let-7, miR-15/16, 26, 29, 31, 34, 145, 200, 203–205, 223, and 375) collectively have more than expected target genes, and well-known cancer genes contain more than expected miRNA binding sites. In summary, miRNA target site distribution exhibits characteristics of the small-world network. The potential to use this pattern to better understand miRNA function and their oncological roles is discussed.

Protonation-dependent heterogeneity in fluorescent binding sites in sub-fractions of fulvic acid using principle component analysis and two-dimensional correlation spectroscopy

Heterogeneous distributions of proton binding sites within sub-fractions of fulvic acid (FA3–FA13) were investigated by use of synchronous fluorescence spectra (SFS), combined with principle component analysis (PCA) and two-dimensional correlation spectroscopy (2D-COS). Tryptophan-like, fulvic-like and humic-like materials were observed in SFS. Tyrosine-like materials were identified by use of SFS-PCA analysis. Combined information from synchronous-asynchronous maps and dissociation constants (pKa) was used to describe heterogeneity of binding sites for protons within each sub-fraction. Heterogeneous distributions of proton binding sites were observed in fulvic-like, humic-like, tryptophan-like, and tyrosine-like materials of five sub-fractions and even in the single fulvic-like materials in FA3 and tryptophan-like materials in FA9 and FA13. Values of pKa of sub-fractions ranged from 2.20 to 5.29, depending on associated wavelengths in synchronous-asynchronous maps and use of the modified Stern-Volmer equation. The larger values of pKa (4.17–5.29) were established for protein-like materials (including tryptophan-like and tyrosine-like materials) in comparison to those (2.20–3.38) for humic-like and fulvic-like materials in sub-fractions. Sequential variations of 274nm (pKa 4.15–5.29)→360–460nm (pKa 2.78–2.39) for FA5–FA13 revealed that binding of protons to tryptophan-like materials appeared prior to humic-like/fulvic-like materials. In FA9, protons were preferentially binding to tryptophan-like materials than tyrosine-like materials. In FA3, protons were preferentially binding to humic-like materials than fulvic-like materials. Relative differences of values of pKa for fluorescent materials within each sub-fraction were consistent with sequential orders derived from asynchronous maps. Such an integrated approach, SFS-PCA/2D-COS, has superior potential for further applications in exploring complex interactions between dissolved organic matter and contaminants in engineered and natural environments.

Distribution of corticotropin-releasing factor (CRF) receptor binding in the mouse brain using a new, high-affinity radioligand, [125 I]-PD-Sauvagine.

The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.

The Arg-Phe-amide peptide 26RFa/glutamine RF-amide peptide and its receptor: IUPHAR Review 24.

The RFamide neuropeptide 26RFa was first isolated from the brain of the European green frog on the basis of cross-reactivity with antibodies raised against bovine neuropeptide FF (NPFF). 26RFa and its N-terminally extended form glutamine RF-amide peptide (QRFP) have been identified as cognate ligands of the former orphan receptor GPR103, now renamed glutamine RF-amide peptide receptor (QRFP receptor). The 26RFa/QRFP precursor has been characterized in various mammalian and non-mammalian species. In the brain of mammals, including humans, 26RFa/QRFP mRNA is almost exclusively expressed in hypothalamic nuclei. The 26RFa/QRFP transcript is also present in various organs especially in endocrine glands. While humans express only one QRFP receptor, two isoforms are present in rodents. The QRFP receptor genes are widely expressed in the CNS and in peripheral tissues, notably in bone, heart, kidney, pancreas and testis. Structure-activity relationship studies have led to the identification of low MW peptidergic agonists and antagonists of QRFP receptor. Concurrently, several selective non-peptidic antagonists have been designed from high-throughput screening hit optimization. Consistent with the widespread distribution of QRFP receptor mRNA and 26RFa binding sites, 26RFa/QRFP exerts a large range of biological activities, notably in the control of energy homeostasis, bone formation and nociception that are mediated by QRFP receptor or NPFF2. The present report reviews the current knowledge concerning the 26RFa/QRFP-QRFP receptor system and discusses the potential use of selective QRFP receptor ligands for therapeutic applications.

The Effect of Metal Concentration on the Parameters Derived from Complexometric Titrations of Trace Elements in Seawater—A Model Study

In this study we examine the impact of dissolved metal concentrations on the parameters that are commonly determined from complexometric titrations in seawater. We use the non-ideal competitive adsorption (NICA) model within the framework of the chemical speciation program visual MINTEQ with iron as a model metal. We demonstrate that dissolved iron concentrations effect the determined parameters for a heterogeneous binding site distribution with a fixed concentration of dissolved organic carbon. The commonly used terms ‘ligand concentration’ and ‘binding constant’ are therefore dependent on metal concentration, so we adopt the terminology suggested by Town and Filella (Limnology and Oceanography, 45, 1341-1357, 2000) and use the terms ligand quotient and stability quotient here. The systematic increase in the ligand quotient with dissolved iron concentration likely contributes towards the trend of increasing ligand quotient with dissolved iron concentration observed in field studies, and makes it hard to assign an objective meaning to the parameter. We suggest that calculation of the side reaction coefficient, a parameter that describes the probability that any added metal will be complexed, could be less prone to bias and misinterpretation than calculation of conditional stability and ligand quotients. We explore the impact of experimental design on side reaction coefficients by applying different detection windows, and multiwindow and reverse titration approaches. We identify the method that results in the best estimates of side reaction coefficients over a range of iron concentrations between 0.1 and 1.5 nmol L-1. We find that single window titrations can only reliably estimate side reaction coefficients over a limited range of iron concentrations. Multiwindow titrations provided estimates of side reaction coefficients within the 99 % confidence interval of the values calculated directly from the NICA model at all iron concentrations examined here. We recommend that future reports of speciation measurements consider the potential influence of metal concentrations on the determined parameters and future studies focus on developing and applying experimental designs that improve the robustness and rigor of chemical speciation analysis in the marine environment.

The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random manner.

Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase or, vice versa, competing with inhibitors to activate transcription.

Abstract 3877: Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma tumors

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 [P3F] or PAX7-FOXO1 [P7F] resulting from 2;13 and 1;13 chromosomal translocations) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. To further examine the significance of DNA methylation, we generated genome-wide DNA methylation profiles for a new cohort of 48 RMS tumors for which we previously assessed mutation, copy number and expression status. Investigation of the RMS subsets defined by methylation clustering revealed a significant association of methylation with P3F versus P7F fusion status in the FP subset, and an association of methylation with RAS mutation status in the FN subset. Localization studies of differentially methylated probes showed these probes were not evenly distributed with respect to annotated genomic features. In particular, hypomethylated probes were enriched in FP tumors in the promoter region and in the intergenic region, whereas hypermethylated probes were enriched in these regions in FN tumors. In contrast, hypermethylated probes were enriched in FP tumors in the 3' UTR region whereas hypomethylated probes were enriched in FN tumors in these regions. In our new larger cohort of cases, there was a significant difference in the distribution of P3F binding sites between genes with and without differential methylation. Integrative analysis of P3F binding sites, promoter methylation and gene expression demonstrated that genes with P3F binding sites tended to be more highly expressed in FP tumors (compared to FN tumors) than genes without P3F binding sites regardless of promoter methylation status. Though promoter hypomethylation is most highly associated with enhanced expression among genes with P3F binding sites, the group of genes with P3F binding sites and promoter hypomethylation is small in number compared to the much larger group of genes with P3F binding sites but without promoter hypomethylation. In conclusion, these results demonstrate the interaction of these epigenetic changes with mutational alterations and transcriptional organization in RMS tumors and provide a direction for future studies of these epigenetic events. Citation Format: Wenyue Sun, Bishwanath Chatterjee, Jack F. Shern, Sivasish Sindiri, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2017-3877

Comprehensive CircRNA expression profile and selection of key CircRNAs during priming phase of rat liver regeneration

BackgroundRat liver regeneration (LR) proceeds along a process of highly organized and ordered tissue growth in response to the loss or injury of liver tissue, during which many physiological processes may play important roles. The molecular mechanism of hepatocyte proliferation, energy metabolism and substance metabolism during rat LR had been elucidated. Further, the correlation of circular RNA (circRNA) abundance with proliferation has recently been clarified. However, the regulatory capacity of circRNA in rat LR remains a fascinating topic.ResultsTo investigate the regulatory mechanism of circRNA during priming phase of rat LR, high-throughput RNA sequencing technology was performed to unbiasedly profile the expression of circRNA during priming phase of rat LR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis was conducted to predict the functions of differentially expressed circRNAs and their host linear transcripts. Co-expression networks of circRNA-miRNA were constructed based on the correlation analysis between the differentially expressed LR-related circRNAs and the condition of their miRNA binding sites. To excavate the key circRNAs in the early phase of rat LR, we comprehensively evaluated and integrated the relationship of expression level between the circRNAs and the linear transcripts as well as the distribution of miRNA binding sites in circRNA sequences.ConclusionsThis paper is the first to employ the comprehensive circRNA expression profile and to investigate circRNA-miRNA interactions during priming phase of rat LR. Two thousand four hundred twelve circRNAs were detected, and 159 circRNAs deriving from 116 host linear transcripts differentially expressed (p < 0.05). Six significantly changed circRNAs during priming phase of rat LR were screened as key circle molecules, and then were validated by qRT-PCR. This study will lay the foundation for revealing the functional roles of circRNAs during rat LR and help solve the remaining clinical problems.

Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter.

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

Genomic binding of PAX8-PPARG fusion protein regulates cancer-related pathways and alters the immune landscape of thyroid cancer.

PAX8-PPARG fusion protein (PPFP) results from a t(2;3)(q13;p25) chromosomal translocation, is found in 30% of follicular thyroid carcinomas, and demonstrates oncogenic capacity in transgenic mice. A PPARG ligand, pioglitazone, is highly therapeutic in mice with PPFP thyroid cancer. However, only limited data exist to characterize the binding sites and oncogenic function of PPFP, or to explain the observed therapeutic effect of pioglitazone. Here we used our previously characterized transgenic mouse model of PPFP follicular thyroid carcinoma to identify PPFP binding sites in vivo using ChIP-seq, and to distinguish genes and pathways regulated directly or indirectly by PPFP with and without pioglitazone treatment via integration with RNA-seq data. PPFP bound to DNA regions containing the PAX8 and/or the PPARG motif, near genes involved in lipid metabolism, the cell cycle, apoptosis, and cell motility; the binding site distribution was highly concordant with our previous study in a rat PCCL3 cell line. Most strikingly, pioglitazone induced an immune cell infiltration including macrophages and T cells only in the presence of PPFP, which may be central to its therapeutic effect.

Using fluorescence quenching combined with two-dimensional correlation fluorescence spectroscopy to characterise the binding-site heterogeneity of dissolved organic matter with copper and mercury in lake sediments

Environmental contextDissolved organic matter (DOM) plays an important role in influencing the migration and ultimate fate of metals. Different sources of pollution resulted in changes in the structure of sediment DOM in Lake Wuliangsuhai. We investigate the binding properties of DOM with CuII and HgII using fluorescence quenching combined with two-dimensional correlation spectroscopy (2D-COS) in order to demonstrate the influence of different sources of DOM on metals. AbstractDissolved organic matter (DOM) was collected from three sampling sites (L1, L2 and L3) in Lake Wuliangsuhai. L1 received upstream industrial wastewater and domestic sewage. L2 had suffered from agricultural non-point source pollution. L3 was situated in the lake outlet area. The complexation of DOM with CuII and HgII was investigated based on fluorescence quenching of the synchronous fluorescence spectra on the addition of CuII and on two-dimensional correlation spectroscopy (2D-COS). The synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of CuII and HgII binding sites, which was not readily detected using only the synchronous fluorescence spectra. CuII and HgII complexation was stronger at shorter wavelengths than at longer wavelengths. Moreover, fluorescence quenching also occurred intensely in the fulvic-like regions (363nm for DOM-Cu in L2 and 365nm for DOM-Hg in L1). The logarithms of the stability constants (log KM) ranged from 4.42 to 6.23, from 4.75 to 4.86, and from 4.80 to 5.73 for DOM-Cu in L1, L2 and L3, respectively, depending on the wavelength. DOM at the longer wavelengths exhibited a higher log KM than that at the shorter wavelengths, and the f values in the protein-like region were clearly high. These results suggest that the influence of the structural and chemical properties of DOM on CuII binding may differ for DOM from different sources. The combined approach of fluorescence quenching and 2D-COS could be applied as a tool for evaluating the metal binding site heterogeneity of DOM.

Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.

Differentiating Defect and Basal Plane Contributions to the Surface Energy of Graphite Using Inverse Gas Chromatography

Historically, reported values for the surface energy of graphite have covered a very wide range. Here, we use finite-dilution inverse gas chromatography (FD-IGC) to show that the dispersive component of the surface energy of graphite has contributions from edge and basal plane defects as well as from the hexagonal carbon lattice. The surface energy associated with the defect-free hexagonal lattice is measured at high probe-coverage to be 63 ± 7 mJ/m2, independent of graphite type. However, the surface energy measured at low probe coverage varied from 125 to 175 mJ/m2 depending on the graphite type. Simulation of the FD-IGC output for different binding site distributions allows us to associate this low-coverage surface energy with the binding of probe molecules to high energy defect sites. Importantly, we find the rate of decay of surface energy with probe coverage to carry information about the defect density. By analyzing the dependence of these properties on flake size, it is possible to separate out th...