Visualizing Epigenetics: Current Advances and Advantages in HDAC PET Imaging Techniques

Abnormal gene regulation as a consequence of flawed epigenetic mechanisms may be central to the initiation and persistence of many human diseases. However, the association of epigenetic dysfunction with disease and the development of therapeutic agents for treatment are slow. Developing new methodologies used to visualize chromatin-modifying enzymes and their function in the human brain would be valuable for the diagnosis of brain disorders and drug discovery. We provide an overview of current invasive and noninvasive techniques for measuring expression and functions of chromatin-modifying enzymes in the brain, emphasizing tools applicable to histone deacetylase (HDAC) enzymes as a leading example. The majority of current techniques are invasive and difficult to translate to what is happening within a human brain in vivo. However, recent progress in molecular imaging provides new, noninvasive ways to visualize epigenetics in the human brain. Neuroimaging tool development presents a unique set of challenges in order to identify and validate CNS radiotracers for HDACs and other histone-modifying enzymes. We summarize advances in the effort to image HDACs and HDAC inhibitory effects in the brain using positron emission tomography (PET) and highlight generalizable techniques that can be adapted to investigate other specific components of epigenetic machinery. Translational tools like neuroimaging by PET and magnetic resonance imaging provide the best way to link our current understanding of epigenetic changes with in vivo function in normal and diseased brains. These tools will be a critical addition to ex vivo methods to evaluate – and intervene – in CNS dysfunction.Chemistry and Chemical Biolog

Calculating and visualizing the density of states for simple quantum mechanical systems

We present a graphical approach to understanding the degeneracy, density of states, and cumulative state number for some simple quantum systems. By taking advantage of basic computing operations, we define a straightforward procedure for determining the relationship between discrete quantum energy levels and the corresponding density of states and cumulative level number. The density of states for a particle in a rigid box of various shapes and dimensions is examined and graphed. It is seen that the dimension of the box, rather than its shape, is the most important feature. In addition, we look at the density of states for a multi-particle system of identical bosons built on the single-particle spectra of those boxes. A simple model is used to explain how the N-particle density of states arises from the single particle system it is based on

Three Dimensional Quantification of Angiotensin II-Induced Murine Abdominal Aortic Aneurysms Using High Frequency Ultrasound

Abdominal aortic aneurysms (AAAs), a localized dilation of the vessel wall of 50% or more above normal, claims approximately 14,000 U.S. lives yearly due to aortic rupture. This commonly asymptomatic disease can only be treated by endovascular stent grafts or invasive surgery, usually after the AAA diameter reaches 5 cm. Because these treatment methods carry serious risk, stem cell therapy is being explored in order to provide a low risk option for managing smaller AAAs. To determine if stem cell therapy, once administered, could stabilize or reduce AAA growth, baseline 3D ultrasound measurements in a control group were first needed. High frequency ultrasound was used on apolipoprotein E-deficient (apoE-/-) mice given angiotensin II (AngII) from subcutaneously implanted osmotic mini pumps. This mouse model developed dissecting AAAs, containing a false and true lumen, which were clearly visualized and quantified using 3D ultrasound imaging. With this ultrasound technique, we found that aneurysm diameter, total volume, and false lumen volume all increased steadily over a period of 28 days once AAAs formed. These data suggest our noninvasive, 3D ultrasound technique can be used to monitor the progression of aneurysms that may be delayed once stem cell therapy is administered

Design, Synthesis, and Evaluation of Hydroxamic Acid-Based Molecular Probes for In Vivo Imaging of Histone Deacetylase (HDAC) in Brain

Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging.Chemistry and Chemical Biolog

Nonlinear Optical Microscopy of Murine Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is a cardiovascular disease characterized by dilation and weakening of the vessel wall. AAA rupture is responsible for approximately 14,000 deaths annually in the United States [1]. Nonlinear optical (NLO) microscopy presents new possibilities for analyzing AAA tissue samples from murine models. Common NLO techniques are two-photon excitation fluorescence (TPEF), which detects the intrinsic autofluorescent properties of elastin, and second-harmonic generation (SHG), which is specific for collagen fibrils. Elastin and collagen, two major extracellular matrix components, help the aortic wall withstand internal pressure. Murine AAAs were created through 1) subcutaneous continuous systemic infusion of angiotensin II (AngII) in hyperlipidemic apolipoprotein E-deficient mice and 2) by intraluminal infusion of elastase (low 0.5 U/ml and high 25 U/ml concentrations) into the infrarenal aorta of rats [2]. We imaged aneurysmal and control tissue using TPEF and SHG and compared the resulting images to sections stained with standard elastin and collagen markers. TPEF images revealed disorganized elastin sheets and SHG images indicated collagen turnover after aneurysm formation. We quantified the relative degree of elastin degradation and collagen content in the aortic media within a user-defined area on sections stained with Verhoeff-van Gieson (VVG) or Masson’s trichrome (MTC), as well as on TPEF and SHG images. Our analysis with VVG-stained sections shows that elastin content in AAA tissue is significantly decreased by 64% in AngII models (P=0.02), by 34% in low concentration elastase models (P=0.07), and by 99% in high concentration elastase models (P=0.03), relative to control aortic tissue

Exon expression arrays as a tool to identify new cancer genes

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples. Principal Findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs). Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes

Electrochemical Insight into the Brust-Schiffrin Synthesis of Au Nanoparticles

The mechanism of the Brust–Schiffrin gold nanoparticle synthesis has been investigated through the use of ion transfer voltammetry at the water/1,2-dichloroethane (DCE) solution interface, combined with X-ray absorption fine structure (XAFS) of the reaction between [AuCl4]− and thiol (RSH) in homogeneous toluene (TL) solution. Ion transfer calculations indicate the formation of [AuCl2]− at RSH/Au ratios from 0.2–2 with a time-dependent variation observed over several days. At RSH/Au ratios above 2 and after time periods greater than 24 h, the formation of Au(I)SR is also observed. The relative concentrations of reaction products observed at the liquid/liquid interface are in excellent agreement with those observed by XAFS for the corresponding reaction in a single homogeneous phase. BH4– ion transfer reactions between water and DCE indicate that the reduction of [AuCl4]− or [AuCl2]− to Au nanoparticles by BH4– proceeds in the bulk organic phase. On the other hand, BH4– was unable to reduce the insoluble [Au(I)SR]n species to Au nanoparticles. The number and size of the nanoparticles formed was dependent on the concentration ratio of RSH/Au, as well as the experimental duration because of the competing formation of the [Au(I)SR]n precipitate. Higher concentrations of nanoparticles, with diameters of 1.0–1.5 nm, were formed at RSH/Au ratios from 1 to 2

Sloan Digital Sky Survey Imaging of Low Galactic Latitude Fields: Technical Summary and Data Release

The Sloan Digital Sky Survey (SDSS) mosaic camera and telescope have obtained five-band optical-wavelength imaging near the Galactic plane outside of the nominal survey boundaries. These additional data were obtained during commissioning and subsequent testing of the SDSS observing system, and they provide unique wide-area imaging data in regions of high obscuration and star formation, including numerous young stellar objects, Herbig-Haro objects and young star clusters. Because these data are outside the Survey regions in the Galactic caps, they are not part of the standard SDSS data releases. This paper presents imaging data for 832 square degrees of sky (including repeats), in the star-forming regions of Orion, Taurus, and Cygnus. About 470 square degrees are now released to the public, with the remainder to follow at the time of SDSS Data Release 4. The public data in Orion include the star-forming region NGC 2068/NGC 2071/HH24 and a large part of Barnard's loop.Comment: 31 pages, 9 figures (3 missing to save space), accepted by AJ, in press, see http://photo.astro.princeton.edu/oriondatarelease for data and paper with all figure

Original Article Design, synthesis, and evaluation of hydroxamic acid-based molecular probes for in vivo imaging of histone deacetylase (HDAC) in brain

Abstract: Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging