Histone Deacetylase Inhibition Induces Apoptosis and Cell Cycle Dysregulation in Human and Murine Cancer Cell Lines

Carcinogenesis is a complex multistep process that requires tumor cells to grow rapidly while overcoming growth inhibitory signals and sustained challenges from the host immune response. Mutations within promoter or enhancer regions, along with epigenetic changes, can induce aberrant expression of genes that regulate differentiation, cell cycle, and apoptosis, all of which enhance potential for cellular transformation. In recent years, our understanding of the biological processes that influence the activation and repression of transcription have evolved to highlight the role of chromatin architecture, and how chromatin remodeling may be utilized for the potential therapeutic benefit of genetic disease. Histone deacetylase inhibitors (HDACi) are small molecule drugs that affect the balance between acetylation and deacetylation of proteins, ultimately influencing cellular processes including gene transcription. There are currently three FDA-approved HDACi on the market (FK228, SAHA, Panobinostat) for cancer treatment, all of which have high systemic toxicity. Therefore, there is need for development of less toxic HDACi with improved tumor specificity. HDACi can exert anti-tumor effects by inducing transcriptional changes in tumor suppressors through modulating acetylation of histones and/or transcription factors. We hypothesize that the high systemic toxicity of the current HDACi is due to their non-selective HDAC activity and that more class-specific HDACi would result in less potent effects in cellular proliferation, death, and off target activity. The present studies tested this hypothesis by comparing effects of multiple HDACi on cell cycle progression and cell death in the murine breast 4T1, human prostate DU145, and human myelomonocytic U937 cancer cell lines. Systemic toxicity was assessed ex vivo using primary murine leukocyte populations. Further, cytotoxic effects of HDACi were tested in myeloid derived suppressor cell populations harvested from 4T1 tumor – bearing mice. Using flow cytometry and fluorescence microscopy, we demonstrate that FK228 and Panobinostat induced apoptosis and cell cycle dysregulation of cancer cell populations in vitro. Ongoing studies are elucidating the mechanisms of cell death and determining the off target effects of these HDACi

Monitoring A Potentially Stressful Situation In Captive Western Lowland Gorillas (gorilla Gorilla Gorilla) Through Analysis Of Behavior and Urinary Cortisol

The concept of quantifying animal welfare has received much discussion, in various industries such as agriculture, laboratory, and zoological facilities. Behavioral, physical, and physiological indicators of welfare have previously been used to assess animal welfare; each having advantages and disadvantages, ranging from the practicality of data collection, to the validity of the data and how it is interpreted. Concurrent assessment of multiple measures is a more robust way to examine animal welfare, which utilizes the advantages of each measure, and provides additional information on which to base conclusions and animal care management decisions. This study used measures of behavior and urinary cortisol to examine the potential stress response of a captive gorilla group to short-term space restriction associated with temporary confinement to indoor housing facilities. The study duration was three months; one month of baseline data collection, one month of indoor restriction, and one month of monitoring post-restriction. All-occurrences of selected behaviors were collected, with an emphasis on social and stress-related behaviors, and urine samples were collected daily from a sub-set of the group. A urinary cortisol metabolite enzyme immunoassay was validated and used to monitor adrenal activity in gorillas. Measured cortisol increases in response to a known stressor (medical illness) provided a physical validation of the cortisol EIA and established biological relevance of the assay system. No significant differences in social behaviors (aggression, affiliation) or stereotypic behaviors were observed. Significant (p\u3c0.05) increases in cortisol concentration were measured, suggesting that the gorillas were responding to a stressor during the study period. The observed cortisol increase was not likely to have been caused exclusively by the temporary indoor confinement. Potential additional causes of increased adrenal activity during the study included: presence of the observer and novelty of re-landscaped outdoor enclosure. While the increases in cortisol concentration demonstrate an observed stress response, the magnitude of this stressor, and thus the degree of the stress response, was minor. The stress experienced was not significant enough to alter the normal biological function of the gorillas, and thus, can be considered negligible. The gorillas\u27 ability to effectively deal with this expected stressor may have been enhanced by the additional enrichment provided to the gorillas during their indoor confinement. Gorillas were provided with additional browse, more enrichment items, additional training sessions, and increased keeper interaction while they remained indoors. These animal care and management techniques may have buffered the predicted negative impact on animal welfare due to increases in stress by providing stimulating novelty in the gorillas\u27 indoor environment

An excess electron bound to urea. I. Canonical and zwitterionic tautomers

The possibility of electron binding to urea was studied at the coupled cluster level of theory with single, double, and noniterative triple excitations. It was found that none of the urea isomers forms a valence anionic state although almost all of them can attach an excess electron and form a stable dipole-bound or Rydberg anion. Moreover, the canonical tautomers are the lowest energy structures of the neutral and anion. The zwitterionic isomer was found to be locally stable only when solvated with an ''extra'' electron and the corresponding anion is a Rydberg species perturbed by a neighboring negative charge

Fusion of morphological data obtained by coronary computed tomography angiography with quantitative echocardiographic data on regional myocardial function

Background: Three-dimensional (3D) fusion of morphological data obtained by coronary computed tomography angiography (CCTA) with functional data from resting and stress echocardiography could potentially provide additional information compared to examination results analyzed separately and increase the diagnostic and prognostic value of non-invasive imaging in patients with suspected coronary artery disease (CAD). Using vendor-independent software developed in our institution, we aimed to assess the feasibility and reproducibility of 3D fusion of morphological CCTA data with echocardiographic data regarding regional myocardial function. Methods: Thirty patients with suspected CAD underwent CCTA and resting transthoracic echocardiography. From CCTA we obtained 3D reconstructions of coronary arteries and left ventricle (LV). Offline speckle-tracking analysis of the echocardiographic images provided parametric maps depicting myocardial longitudinal strain in 17 segments of the LV. Using our software, 3 independent investigators fused echocardiographic maps with CCTA reconstruc­tions in all patients. Based on the obtained fused models, each segment of the LV was assigned to one of the major coronary artery branches. Results: Mean time necessary for data fusion was 65 ± 7 s. Complete agreement between independent investigators in assignment of LV segments to coronary branches was obtained in 94% of the segments. The average coefficient of agreement (kappa) between the investigators was 0.950 and the intra-class correlation coefficient was 0.9329 (95% CI 0.9227–0.9420). Conclusions: Three-dimensional fusion of morphological CCTA data with quantitative echocardiographic data on regional myocardial function is feasible and allows highly repro­ducible assignment of myocardial segments to coronary artery branches

A Proline-Based Neuraminidase Inhibitor: DFT Studies on the Zwitterion Conformation, Stability and Formation

The designs of potent neuraminidase (NA) inhibitors are an efficient way to deal with the recent “2009 H1N1” influenza epidemic. In this work, density functional calculations were employed to study the conformation, stability and formation of the zwitterions of 5-[(1R,2S)-1-(acetylamino)-2-methoxy-2-methylpentyl]-4-[(1Z)-1-propenyl]-(4S,5R)-d-proline (BL), a proline-based NA inhibitor. Compared to proline, the zwitterion stability of BL is enhanced by 1.76 kcal mol−1 due to the introduction of functional groups. However, the zwitterion of BL will not represent a local minimum on the potential energy surface until the number of water molecules increases up to two (n = 2). With the addition of two and three water molecules, the energy differences between the zwitterions and corresponding canonical isomers were calculated at 3.13 and −1.54 kcal mol−1, respectively. The zwitterions of BL are mainly stabilized by the H-bonds with the water molecules, especially in the case of three water molecules where the carboxyl-O atoms are largely coordination-saturated by three H-bonds of medium strengths, causing the zwitterion stability even superior to the canonical isomer. With the presence of two and three water molecules, the energy barriers for the conversion processes from the canonical isomers to the zwitterions are equal to 4.96 and 3.13 kcal mol−1, respectively. It indicated that the zwitterion formation is facile to take place with addition of two molecules and further facilitated by more water molecules. Besides, the zwitterion formation of BL is finished in a single step, different from other NA inhibitors. Owing to the above advantages, BL is a good NA inhibitor candidate and more attention should be paid to explorations of BL-based drugs

Hydrogen Bonding Constrains Free Radical Reaction Dynamics at Serine and Threonine Residues in Peptides

Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free-radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal C_α–C or N–C_α bond rather than the typical Cα–C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side-chain hydroxyl group with a backbone carbonyl oxygen aligns the singly occupied π orbital on the β-carbon and the N–C_α bond, leading to low-barrier β-cleavage of the N–C_α bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z• ions, whereas C-terminal interaction leads to effective cleavage of the C_α–C bond through rapid loss of isocyanic acid. Dissociation of the C_α–C bond may also occur via water loss followed by β-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes