Methods for accurately quantitating changes in histone post-translational modifications are necessary for developing an understanding of how their dynamic nature influences nuclear events involving ...access to genomic DNA. This article describes methods for the use of in vivo stable isotope label incorporation for quantitating the levels of modification at specific residues in histone proteins. These methods are applicable to a wide variety of model systems and examples of their use in both mammalian cells and
Saccharomyces cerevisiae are presented.
Abstract only Background: Clinical data support that inflammation and the improper regulation of the immune response are intimately associated with Heart Failure (HF), however, the type of immune ...response involved and whether it regulates cardiac remodeling remains largely unexplored. We hypothesize that T cell mediated immune responses and their recruitment into the heart influence cardiac remodeling and contribute to the pathogenesis of pressure overload induced HF. Methods and Results: Using quantitative flow cytometry we found that T cells infiltrated the heart as Wild-type mice (WT) developed systolic dysfunction and LV hypertrophy in response to transverse aortic constriction (TAC) (p<0.01 TAC vs Sham). Real time imaging demonstrated that T cells from TAC mice adhered to activated heart endothelial cells in higher numbers than T cells from Sham mice under physiological flow conditions in vitro (P<0.05) indicating a systemic T cell activation to pressure overload induced by TAC. Similarly, circulating T cells from patients with HF adhered more to activated human umbilical vein endothelial cells (HUVEC) than T cells from healthy volunteers. Based on these findings, we performed similar TAC studies in T cell deficient mice (TCRα -/- ). In contrast with WT TAC mice, TCRα -/- had preserved LV systolic and diastolic function (p<0.01) determined by echocardiography and hemodynamic studies, reduced LV fibrosis (p<0.001) and TGFβ1, collagen Iα and αSMA gene expression (p<0.05), and reduced LV hypertrophy and gene expression of ANP and BNP (p<0.05), but unaltered expression of SerCA. Remarkably, TCRα -/- had improved survival after 4 weeks of TAC 100%(16/16) TCRα -/- vs 73.7%(14/19) WT, p=0.023. Ongoing studies will determine the mechanisms regulating T cell recruitment into the heart, the type of T cell response involved and its contribution to pathological remodeling of the heart. Conclusion: Our studies demonstrate that T cell immune responses and their recruitment into the LV contribute to the pathogenesis of pressure overload induced HF by mechanisms involving T cell regulation of cardiac hypertrophy and fibrosis, and open a window to develop novel therapeutic strategies to improve the structural, functional and molecular deficits of the failing heart.
Novel analogue
5 of naltrexone displays very high affinity for opioid receptors with
K
i values of 0.052
nM, 2.6
nM and 0.23
nM for μ, δ and κ, respectively.
Very high affinity for opioid receptors ...(e.g.,
K
i
=
0.052
nM for μ) has been observed in the rationally designed naltrexone analogue
5. SAR and physical data supports the hypothesis that the 4-OH group of
5 stabilizes the 3-carboxamido group in the putative bioactive conformation.
A combination of conventional organic synthesis, remotely monitored flow synthesis and bioassay platforms, were used for the evaluation of novel inhibitors targeting bromodomains outside the ...well-studied bromodomain and extra terminal (BET) family, here exemplified by activity measurements on the bromodomain of BRD9 protein, a component of some tissue-specific SWi/SNF chromatin remodelling complexes. The Frontal Affinity Chromatography combined with Mass Spectrometry (FAC-MS) method proved to be reliable and results correlated well with an independent thermal shift assay.
The recent disclosure of type I1/2 inhibitors for p38 MAPK demonstrated how the stabilization of the R-spine can be used as a strategy to greatly increase the target residence time (TRT) of ...inhibitors. Herein, we describe first time n-acylhydrazone and selenophene residues as spine-motives, yielding metabolically stable inhibitors with high potency on enzymatic, Nano-BRET and whole blood assays, improved metabolic stability and long TRT.
Objective. To describe the pharmacokinetics of maraviroc in human immunodeficiency virus (HIV)–infected women during pregnancy and post partum. Methods. HIV-infected pregnant women receiving ...maraviroc as part of clinical care had intensive steady-state 12-hour pharmacokinetic profiles performed during the third trimester and ≥2 weeks after delivery. Cord blood samples and matching maternal blood samples were taken at delivery. The data were collected in 2 studies: P1026 (United States) and PANNA (Europe). Pharmacokinetic parameters were calculated. Results. Eighteen women were included in the analysis. Most women (12; 67%) received 150 mg of maraviroc twice daily with a protease inhibitor, 2 (11%) received 300 mg twice daily without a protease inhibitor, and 4 (22%) had an alternative regimen. The geometric mean ratios for third-trimester versus postpartum maraviroc were 0.72 (90% confidence interval, .60–.88) for the area under the curve over a dosing interval (AUCtau) and 0.70 (0.58–0.85) for the maximum maraviroc concentration. Only 1 patient showed a trough concentration (Ctrough) below the suggested target of 50 ng/mL, both during pregnancy and post partum. The median ratio of maraviroc cord blood to maternal blood was 0.33 (range, 0.03–0.56). The viral load close to delivery was <50 copies/mL in 13 women (76%). All children were HIV negative at testing. Conclusions. Overall maraviroc exposure during pregnancy was decreased, with a reduction in AUCtau and maximum concentration of about 30%. Ctrough was reduced by 15% but exceeded the minimum Ctrough target concentration. Therefore, the standard adult dose seems sufficient in pregnancy. Clinical Trials Registration. NCT00825929 and NCT000422890.
Corticotrophinomas, which are neuroendocrine tumors (NETs) and represent >10% of all surgically removed pituitary adenomas, cause Cushing’s disease that is associated with hypersecretion of ...adrenocorticotropic hormone (ACTH) leading to excessive production of glucocorticoids by the adrenal cortex. Trans-sphenoidal hypophysectomy is the treatment of choice for corticotrophinomas, with medical treatments being reserved for patients who have contraindications for surgery. However, these treatments are often ineffective. Improved therapeutic approaches are required, and we have therefore assessed the efficacy of epigenetic modulators, a new class of anti-cancer drugs that have been reported to be effective in treating pancreatic NETs. Specifically, we assessed the anti-proliferative and pro-apoptotic effects of the bromo and extra terminal domain (BET) inhibitors JQ1 and PFI-1, in the mouse corticotrophinoma cell line AtT20, using Cell Titer Blue and Caspase Glo assays, respectively. JQ1, after 96h treatment, was more efficacious than PFI-1. Thus, JQ1 significantly decreased proliferation by 95%, (p<0.0005), and significantly increased apoptosis >50-fold (p<0.0005), compared to control treated cells; whereas PFI-1 decreased proliferation by 43% (p<0.0005), but did not significantly alter apoptosis. Furthermore, AtT20 cells did not resume proliferating for up to 96 hours after the removal of JQ1 from the media. In addition, RNA-sequence (RNA-Seq) analysis revealed that JQ1 treatment significantly altered the expression of genes involved in apoptosis, including Nuclear Factor Kappa B Subunit 1 (
Nfκb1
) and Baculoviral IAP Repeat Containing 3 (
Birc3
), as well as genes associated with the somatostatin receptor type 2 (SSTR2) anti-proliferative signaling pathway, including
Sstr2
. The down regulation of these genes was confirmed using quantitative PCR, which showed decreases in
Nfkb1
,
Birc3
and
Sstr2
mRNA of 2.9-fold (p<0.0009), 1.7-fold (p<0.005), and 1.5-fold (p<0.005), respectively. In addition, Western blot analysis showed decreases in Nfkb1, cIAP2 and SSTR2 protein expression of 1.5-fold (p<0.05), 1.9-fold (p<0.05), and 2.3-fold (p<0.05), respectively. Thus our results, which demonstrate that JQ1 treatment is both anti-proliferative and pro-apoptotic in ACTH-secreting cells, reveal that BET inhibition may provide a novel approach for the treatment of corticotrophinomas.
Ecological intensification has been embraced with great interest by the academic sector but is still rarely taken up by farmers because monitoring the state of different ecological functions is not ...straightforward. Modelling tools can represent a more accessible alternative of measuring ecological functions, which could help promote their use amongst farmers and other decision-makers. In the case of crop pollination, modelling has traditionally followed either a mechanistic or a data-driven approach. Mechanistic models simulate the habitat preferences and foraging behaviour of pollinators, while data-driven models associate georeferenced variables with real observations. Here, we test these two approaches to predict pollination supply and validate these predictions using data from a newly released global dataset on pollinator visitation rates to different crops. We use one of the most extensively used models for the mechanistic approach, while for the data-driven approach, we select from among a comprehensive set of state-of-the-art machine-learning models. Moreover, we explore a mixed approach, where data-derived inputs, rather than expert assessment, inform the mechanistic model. We find that, at a global scale, machine-learning models work best, offering a rank correlation coefficient between predictions and observations of pollinator visitation rates of 0.56. In turn, the mechanistic model works moderately well at a global scale for wild bees other than bumblebees. Biomes characterized by temperate or Mediterranean forests show a better agreement between mechanistic model predictions and observations, probably due to more comprehensive ecological knowledge and therefore better parameterization of input variables for these biomes. This study highlights the challenges of transferring input variables across multiple biomes, as expected given the different composition of species in different biomes. Our results provide clear guidance on which pollination supply models perform best at different spatial scales – the first step towards bridging the stakeholder–academia gap in modelling ecosystem service delivery under ecological intensification.
The structural distortion of high-generation poly(amidoamine) (PAMAM) dendrimers at the liquid/solid interface was investigated in situ using tapping mode atomic force microscopy (AFM). With a ...substantial compression along the surface normal but minor lateral expansion, isolated PAMAM dendrimers at the water/mica interface exhibit dimensions similar to those reported for the same species in dense, dried arrays, rather than those of isolated, dried units. On graphite substrates, adsorbed dendrimers exhibit weaker adhesion forces than on mica. AFM images of lower generation dendrimers at the water/graphite interface reveal additional features interpreted as large three-dimensional aggregates.
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