The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Sirt3, Mitochondrial ROS, Ageing, and Carcinogenesis

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis

Nigella sativa (Black Cumin) Seed Extract Alleviates Symptoms of Allergic Diarrhea in Mice, Involving Opioid Receptors

The incidence of food hypersensitivity and food allergies is on the rise and new treatment approaches are needed. We investigated whether N. sativa, one of its components, thymoquinone, or synthetic opioid receptor (OR)-agonists can alleviate food allergy. Hence, ovalbumin (OVA) -sensitized BALB/c-mice were pre-treated either with a hexanic N. sativa seed extract, thymoquinone, kappa- (U50'4889) or mu-OR-agonists (DAMGO) and subsequently challenged intra-gastrically with OVA. All 4 treatments significantly decreased clinical scores of OVA-induced diarrhea. N. sativa seed extract, thymoquinone, and U50'488 also decreased intestinal mast cell numbers and plasma mouse mast cell protease-1 (MMCP-1). DAMGO, in contrast, had no effect on mast cell parameters but decreased IFNγ, IL-4, IL-5, and IL-10 concentration after ex vivo re-stimulation of mesenteric lymphocytes. The effects on allergy symptoms were reversible by OR-antagonist pre-treatment, whereas most of the effects on immunological parameter were not. We demonstrate that N. sativa seed extract significantly improves symptoms and immune parameters in murine OVA-induced allergic diarrhea; this effect is at least partially mediated by thymoquinone. ORs may also be involved and could be a new target for intestinal allergy symptom alleviation. N. sativa seed extract seems to be a promising candidate for nutritional interventions in humans with food allergy

Summary Report of PQRI Workshop on Nanomaterial in Drug Products: Current Experience and Management of Potential Risks

At the Product Quality Research Institute (PQRI) Workshop held last January 14-15, 2014, participants from academia, industry, and governmental agencies involved in the development and regulation of nanomedicines discussed the current state of characterization, formulation development, manufacturing, and nonclinical safety evaluation of nanomaterial-containing drug products for human use. The workshop discussions identified areas where additional understanding of material attributes, absorption, biodistribution, cellular and tissue uptake, and disposition of nanosized particles would continue to inform their safe use in drug products. Analytical techniques and methods used for in vitro characterization and stability testing of formulations containing nanomaterials were discussed, along with their advantages and limitations. Areas where additional regulatory guidance and material characterization standards would help in the development and approval of nanomedicines were explored. Representatives from the US Food and Drug Administration (USFDA), Health Canada, and European Medicines Agency (EMA) presented information about the diversity of nanomaterials in approved and newly developed drug products. USFDA, Health Canada, and EMA regulators discussed the applicability of current regulatory policies in presentations and open discussion. Information contained in several of the recent EMA reflection papers was discussed in detail, along with their scope and intent to enhance scientific understanding about disposition, efficacy, and safety of nanomaterials introduced in vivo and regulatory requirements for testing and market authorization. Opportunities for interaction with regulatory agencies during the lifecycle of nanomedicines were also addressed at the meeting. This is a summary of the workshop presentations and discussions, including considerations for future regulatory guidance on drug products containing nanomaterials

The therapeutic potential of epigenetic manipulation during infectious diseases.

Epigenetic modifications are increasingly recognized as playing an important role in the pathogenesis of infectious diseases. They represent a critical mechanism regulating transcriptional profiles in the immune system that contributes to the cell-type and stimulus specificity of the transcriptional response. Recent data highlight how epigenetic changes impact macrophage functional responses and polarization, influencing the innate immune system through macrophage tolerance and training. In this review we will explore how post-translational modifications of histone tails influence immune function to specific infectious diseases. We will describe how these may influence outcome, highlighting examples derived from responses to acute bacterial pathogens, models of sepsis, maintenance of viral latency and HIV infection. We will discuss how emerging classes of pharmacological agents, developed for use in oncology and other settings, have been applied to models of infectious diseases and their potential to modulate key aspects of the immune response to bacterial infection and HIV therapy

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, “S-XL6,” was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A’s in vivo half-life; and that S-XL6 crosses the blood–brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS

Eukaryotic initiation factor 4B (eIF4B) : regulation by signaling pathways and its role in translation

Due to the high energetic expenditure for the cell, the protein biosynthesis in eukaryotes is an extensively controlled process predominantly regulated at the ribosomal biogenesis and translation initiation steps. The ribosomal biogenesis defines the global translational aptitude of the cell. It is a mainly nucleolar process which is regulated at multiple steps (e.g. transcription, rRNA processing and modification, ribosomal protein translation etc). However, the most extensively regulated and the rate limiting step of translation is the initiation. Multiple eukaryotic translation initiation factors (eIFs) function to facilitate this priming step of translation. The initial recognition of the mRNA molecule happens through the 5' cap structure found in all mRNAs of nuclear origin. This event is mediated through the recruitment of heterotrimeric complex eIF4F consisting of cap-binding protein eIF4E, scaffolding protein eIF4G and the RNA helicase eIF4A unwinding secondary structures found in 5'UTR of mRNA and thus thought to facilitate the scanning process. The helicase activity of elF4F complex or of eIF4A alone is further potentiated by eIF4B in vitro. The latter protein is at the focus of present thesis.Signal transduction regulates multiple cellular processes including mitogenesis, differentiation, apoptosis, chemotaxis etc. Signaling pathways also regulate ribosomal biogenesis to coordinate mitogenic cues, nutrient and energy availability with the translational capacity of the cells. Mounting evidence links PI3K-Akt-mTOR and Ras-MAPK cascades to the translational control. In this thesis, I show that PI3K/mTOR and MAP kinase cascades converge to phosphorylate eIF4B on Ser422. This phosphorylation results in an increased interaction with eIF3, an essential factor bridging between eIF4F and the small ribosomal subunit. Physiological significance of eIF4B phosphorylation on Ser422 has been demonstrated by the stimulatory effect of eIF4B Ser422Asp phosphomimetic mutant on cap-dependent translation. Taken together, this represents a new paradigm of translational control mechanism regulated by signaling crosstalk. The function of eIF4B in vitro is well characterized but its in vivoeffects are disputed in literature. To address this I established HeLa cell line stably expressing shRNA targeting eIF4B. eIF4B silencing inhibits proliferation rates and anchorage-independent growth. Expression of luciferase reporter gene containing 5' terminal oligopyrimidine tract (TOP) is selectively repressed in eIF4B-silenced cells and can be rescued by exogenous eIF4B regardless of Ser422 phosphorylation status. Moreover, the de novo synthesis rates of endogenous ribosomal proteins in serum starved cultures recapitulate the luciferase reporter assay data. Utilizing polysomal analysis, I was able to show more significant inhibition of translation initiation in serum starved eIF4B-silenced cells. Our attempt to discover novel eIF4B-interacting proteins by Mass Spectrometry approach led to the identification of nucleolar RNA helicase DDX21. Confocal microscopy has shown partial co-localization of tagged eIF4B and DDX21 in nucleolar periphery. Pulse chase experiments metabolically labeling rRNA show an attenuated 28S rRNA production and concomitant accumulation of 36S intermediates in eIF4B-silenced cells. Since ribosomal biogenesis is highly coordinated process and requires strict stoichiometry maintenance of ribosomal components the observed inhibition of rRNA processing could be consequential to the decreased ribosomal protein expression. However, given the fact that eIF4B is associated with the nucleolar pre-ribosomal particle complexes its direct effect on rRNA processing cannot be ruled out. Regulation of ribosomal biogenesis by translation initiation factor may represent an important control mechanism allowing cells to co-ordinate these two processes