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A family of mesothermal ionic liquids comprised of tetraarylphosphonium cations and the bis(trifluoromethanesulfonyl)amidate anion are shown to be materials of exceptional thermal stability, enduring (without decomposition) heating in air at 300 °C for three months. It is further established that three specific structural elements - phenoxy, phenacyl, and phenyl sulfonyl - can be present in the cation structures without compromising their thermal stability, and that their incorporation has specific impacts on the melting points of the salts. Most importantly, it is shown that the ability of such a structural component to lower a salt melting point is tied to its ability to lower cation-cation repulsions in the material.

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [ 32 P/ 33 P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

RNA-mediated transcriptional gene silencing is a conserved process where small RNAs target transposons and other sequences for repression by establishing chromatin modifications. A central element of this process are long non-coding RNAs (lncRNA), which in Arabidopsis thaliana are produced by a specialized RNA polymerase known as Pol V. Here we show that non-coding transcription by Pol V is controlled by preexisting chromatin modifications located within the transcribed regions. Most Pol V transcripts are associated with AGO4 but are not sliced by AGO4. Pol V-dependent DNA methylation is established on both strands of DNA and is tightly restricted to Pol V-transcribed regions. This indicates that chromatin modifications are established in close proximity to Pol V. Finally, Pol V transcription is preferentially enriched on edges of silenced transposable elements, where Pol V transcribes into TEs. We propose that Pol V may play an important role in the determination of heterochromatin boundaries., Competing Interests: The authors declare that no competing interests exist.

Upregulation of RNA Polymerase (Pol I)-mediated transcription of rRNA and increased ribogenesis are hallmarks of breast cancer. According to several datasets, including The Cancer Genome Atlas (TCGA), amplification/upregulation of genes encoding for basal components of the Pol I transcriptional machinery is frequent at different breast cancer stages. Here we show that knock down of the RNA polymerase I-specific transcription initiation factor RRN3 (TIF-IA) in breast cancer cells is sufficient to reduce rRNA synthesis and inhibit cell proliferation, and second that stable ectopic expression of RRN3 in human mammary epithelial (HME1) cells, by increasing rRNA transcription, confers increased sensitivity to the anti-proliferative effects of a selective Pol I inhibitor. Further, RRN3-overexpressing HME1 cells, when grown in in vitro 3-dimensional (3D) culture, develop into morphologically aberrant acinar structures lacking a lumen and filled with proliferative cells, thus acquiring a morphology resembling in situ ductal breast cancer lesions (DCIS). Consequently, interference with RRN3 control of Pol I transcription seems capable of both compromising mammary epithelial morphogenetic processes at early breast cancer stages, and driving breast cancer progression by fostering proliferation.

Long noncoding RNA (lncRNA) is a pivotal factor regulating various aspects of genome activity. Genome regulation via DNA methylation and post-translational histone modifications is a well-documented function of lncRNA in plants, fungi, and animals. Here, we summarize evidence showing that lncRNA also controls chromatin structure, including nucleosome positioning and chromosome looping. We focus on data from plant experimental systems, discussed in the context of other eukaryotes. We explain the mechanisms of lncRNA-controlled chromatin remodeling and the implications of the functional interplay between noncoding transcription and several different chromatin remodelers. We propose that the unique properties of RNA make it suitable for controlling chromatin modifications and structure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Previous studies suggest the anti-inflammatory drug, sulindac inhibits tumorigenesis by a COX independent mechanism involving cGMP PDE inhibition. Here we report that the cGMP PDE isozymes, PDE5 and 10, are elevated in colon tumor cells compared with normal colonocytes, and that inhibitors and siRNAs can selectively suppress colon tumor cell growth. Combined treatment with inhibitors or dual knockdown suppresses tumor cell growth to a greater extent than inhibition from either isozyme alone. A novel sulindac derivative, ADT-094 was designed to lack COX-1/-2 inhibitory activity but have improved potency to inhibit PDE5 and 10. ADT-094 displayed >500 fold higher potency to inhibit colon tumor cell growth compared with sulindac by activating cGMP/PKG signaling to suppress proliferation and induce apoptosis. Combined inhibition of PDE5 and 10 by treatment with ADT-094, PDE isozyme-selective inhibitors, or by siRNA knockdown also suppresses β-catenin, TCF transcriptional activity, and the levels of downstream targets, cyclin D1 and survivin. These results suggest that dual inhibition of PDE5 and 10 represents novel strategy for developing potent and selective anticancer drugs.

The remarkable properties of bone derive from a highly organized arrangement of coaligned nanometer-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the nonmineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite. We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and atomic force microscopy (AFM) observations of collagen adsorption on single crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and transmission electron microscopy (TEM) analyses of native tissues shows that only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations, and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular-scale organization of bone.

A series of novel lipid-inspired ionic liquids have been synthesized employing the thiol-ene "click" reaction in a single-step process. The thermal properties were determined by differential scanning calorimetry (DSC) and showed observable trends between the C16, C18, and C20 analogues. The minimum melting points for each equivalent chain length series occur at sequential odd sulfur positions, 3, 5, and 7 for the C16, C18, and C20 series, respectively. The magnitude of melting point depression relative to the saturated homologue is observed to have a strong dependence on the position of the sulfur in the side chain. Additionally, the sulfur position corresponding to the lowest melting point for a homologous series shifts further down the chain as the chain length is increased, indicating that the maximum effect takes place near the center of the ion and not the center of the thiaalkyl chain. This synthesis provides tunability and improved thermal stability for 1-methyl-3-thiaalkylimidazolium bistriflimides and insight into structure-property relationships of lipidic ionic liquids.

Transcriptional gene silencing controls the activity of transposable elements and expression of protein-coding genes. It requires non-coding transcription, which in plants is performed by RNA Polymerases IV and V (Pol IV and Pol V). Pol IV produces precursors for siRNA biogenesis while Pol V produces scaffold transcripts required for siRNAs and associated proteins to recognize their target loci. In this review I discuss the mechanisms used by Pol IV and Pol V to mediate repressive chromatin modifications. I further discuss the mechanisms controlling non-coding transcription and their role in regulation of genome activity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects., (Copyright © 2012 International Society for Advancement of Cytometry.)

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Retrotransposons and repetitive DNA elements in eukaryotes are silenced by small RNA-directed heterochromatin formation. In Arabidopsis, this process involves 24-nt siRNAs that bind to ARGONAUTE4 (AGO4) and facilitate the targeting of complementary loci via unknown mechanisms. Nuclear RNA polymerase V (Pol V) is an RNA silencing enzyme recently shown to generate noncoding transcripts at loci silenced by 24-nt siRNAs. We show that AGO4 physically interacts with these Pol V transcripts and is thereby recruited to the corresponding chromatin. We further show that DEFECTIVE IN MERISTEM SILENCING3 (DMS3), a structural maintenance of chromosomes (SMC) hinge-domain protein, functions in the assembly of Pol V transcription initiation or elongation complexes. Collectively, our data suggest that AGO4 is guided to target loci through base-pairing of associated siRNAs with nascent Pol V transcripts.

Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme required for siRNA-mediated gene silencing of transposons and other repeats, transcribes intergenic and noncoding sequences, thereby facilitating heterochromatin formation and silencing of overlapping and adjacent genes. Pol IVb/Pol V transcription requires the chromatin-remodeling protein DRD1 but is independent of siRNA biogenesis. However, Pol IVb/Pol V transcription and siRNA production are both required to silence transposons, suggesting that Pol IVb/Pol V generates RNAs or chromatin structures that serve as scaffolds for siRNA-mediated heterochromatin-forming complexes. Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region.

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Under near-physiological pH, temperature, and ionic strength, a kinetics constant composition (CC) method was used to examine the roles of phosphorylation of a 14 amino acid segment (DDVDDTDDSHQSDE) corresponding to potential crystal binding domains within the osteopontin (OPN) sequence. The phosphorylated 14-mer OPN peptide segment significantly inhibits both the nucleation and growth of calcium oxalate monohydrate (COM), inhibiting nucleation by markedly increasing induction times and delaying subsequent growth by at least 50% at concentrations less than 44 nM. Molecular modeling predicts that the doubly phosphorylated peptide binds much more strongly to both (-101) and (010) faces of COM. The estimated binding energies are, in part, consistent with the CC experimental observations. Circular dichroism spectroscopy indicates that phosphorylation does not result in conformational changes in the secondary peptide structure, suggesting that the local binding of negatively charged phosphate side chains to crystal faces controls growth inhibition. These in vitro results reveal that the interactions between phosphorylated peptide and COM crystal faces are predominantly electrostatic, further supporting the importance of macromolecules rich in anionic side chains in the inhibition of kidney stone formation. In addition, the phosphorylation-deficient form of this segment fails to inhibit COM crystal growth up to concentrations of 1450 nM. However, at sufficiently high concentrations, this nonphosphorylated segment promotes COM nucleation. Dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) results confirm that aggregation of the nonphosphorylated peptide segment takes place in solution above 900 nM when the aggregated peptide particles may exceed a well-defined minimum size to be effective crystallization promoters.

Objective: Calcium deposition occurs frequently in osteoarthritic (OA) joints. However, evidence for a causal role of calcification in cartilage degeneration is inferential. The present study was undertaken to examine the role of calcification in OA disease progression and to evaluate a formulation of phosphocitrate (PC) as a potential therapeutic agent., Methods: We have identified a guinea pig OA model in which meniscal calcification appears to correlate with aging and disease progression. We synthesized a new formulation of PC, [CaNa(PC)2(H2O)](n) (CaNaPC), which is a potent antimineralization agent and a specific inhibitor of crystal-induced biologic effects. After weekly treatment of guinea pigs with experimental OA with CaNaPC for 3 months, we examined calcification in menisci and cartilage degeneration. As a control, we examined whether similar CaNaPC treatment had any therapeutic effect in a hemi-meniscectomy model in which there is no known crystal involvement., Results: Meniscal calcification correlated with cartilage degeneration in this animal model. PC treatment led to significant reduction of calcium deposits and arrested OA disease progression. Similar treatment had no effect in the hemi-meniscectomy model., Conclusion: CaNaPC diminishes mineralization in a cutaneous calcergy model and a model of OA in which intraarticular mineralization is a prominent feature. In the OA guinea pig model, inhibition of calcification is accompanied by diminished cartilage degeneration. CaNaPC has no therapeutic effect in the hemi-meniscectomy model. We conclude that pathologic calcification may initiate or amplify processes leading to cartilage degeneration and that CaNaPC may interrupt such a pathway.

The majority of human kidney stones are composed primarily of calcium oxalate monohydrate (COM) crystals. Thus, determining the molecular modulation of COM crystallization by urinary constituents is crucial for understanding and controlling renal stone disease. A comprehensive molecular-scale view of COM shape modification by citrate, obtained through a combination of in situ atomic force microscopy and molecular modeling, is presented here. We find that while the most important factors determining binding strength are coordination between COO- groups on citrate and Ca ions in the lattice, as well as H-bonds formed between the OH group of citrate and an oxalate group, the nonplanar geometry of the steps provides the most favorable environment due to the ability of the step-edge to accommodate all Ca-COO- coordinations with minimal strain. However, binding to all steps and terraces on the (010) face is much less favorable than on the (101) face due to electrostatic repulsion between oxalate and COO- groups. For example, the maximum binding energy, -166.5 kJ mol(-1), occurs for the [101] step on the (101) face, while the value for the [021] step on the (010) face is only -56.9 kJ mol(-1). This high selectivity leads to preferential binding to steps on the (101) face that pins step motion. Yet anisotropy in interaction strength on this face drives anisotropic changes in step kinetics that are responsible for shape modification of macroscopic COM crystals. Thus, the molecular scale growth kinetics and the bulk crystal habit are fully consistent with the simulations.

Histone H1 is an abundant component of eukaryotic chromatin that is thought to stabilize higher-order chromatin structures. However, the complete knock-out of H1 genes in several lower eukaryotes has no discernible effect on their appearance or viability. In higher eukaryotes, the presence of many mutually compensating isoforms of this protein has made assessment of the global function of H1 more difficult. We have used double-stranded RNA (dsRNA) silencing to suppress all the H1 genes of Arabidopsis thaliana. Plants with a >90% reduction in H1 expression exhibited a spectrum of aberrant developmental phenotypes, some of them resembling those observed in DNA hypomethylation mutants. In subsequent generations these defects segregated independently of the anti-H1 dsRNA construct. Downregulation of H1 genes did not cause substantial genome-wide DNA hypo- or hypermethylation. However, it was correlated with minor but statistically significant changes in the methylation patterns of repetitive and single-copy sequences, occurring in a stochastic manner. These findings reveal an important and previously unrecognized link between linker histones and specific patterns of DNA methylation.

The aim of this study was to demonstrate iNOS mRNA expression in muscular phase of experimental trichinellosis and to localize iNOS protein in T. spiralis-infected muscles using specific anti-iNOS monoclonal antibodies. The expression of iNOS mRNA in skeletal muscles from Trichinella spiralis-infected mice was examined using the reverse transcription PCR assay. Fragments of skeletal muscles were also subjected to the immunohistochemical reaction using specific anti-iNOS monoclonal antibodies followed by Dako-Ark test. mRNA for iNOS measured on day 21 after infection was expressed in the muscular phase of trichinellosis. Positive immunostaining for iNOS occurred in infiltrating mononuclear cells around the encapsulated larvae. iNOS-positive cells could be traced from the 21st day post infection (dpi); on 42 dpi and 90 dpi most cells expressed iNOS. By assessing expression of protein and its mRNA it can be concluded that iNOS is active in the pathology of skeletal muscle tissue in experimental trichinellosis.

Induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope (env; gp160) glycoprotein has been demonstrated with orally administered recombinant vaccinia virus (rVV) vectors and poly(DL-lactide-co-glycolide) (PLG)-encapsulated plasmid DNA expressing gp160. In this study, we investigated the effect of an oral DNA-prime/rVV-boost vaccine regimen in conjunction with adjuvants on the level of gp160-specific cellular and humoral responses in BALB/c mice. We demonstrated that DNA priming followed by a booster with rVV expressing gp160 (vPE16) significantly augmented env-specific immunity in systemic and mucosal tissues of the immunized mice. Association of vPE16 with liposomes and coadministration of liposome-associated beta-glucan lentinan or IL-2/Ig-encoded plasmid DNA-encapsulated in PLG microparticles triggered the optimal cell-mediated immune (CMI) responses. Lentinan was found to increase env-specific type 1 cytokine production and cytotoxic T-lymphocyte (CTL) activities but had no effect on humoral responses. On the other hand, IL-2/Ig-mediated increases in both type 1 and 2 activities were associated with higher levels of env-specific CTL and antibody responses. Results of these studies demonstrated the effectiveness of oral vaccines with DNA and rVV vectors in conjunction with immunomodulators in inducing specific immune responses in systemic and mucosal tissues.