Developing a Model System to Study the Mechanisms of Resveratrol Inhibition of AR-V7 Transcriptional Activity

Prostate cancer is one of the biggest threats to men’s health in the western world and it accounts for the second largest number of male cancer-related deaths in the United States. It is well established that prostate cancer cells depend on the androgen/androgen receptor pathway. Therefore, androgen deprivation therapy (ADT) has become the primary treatment option for prostate cancer. Patients with metastatic prostate cancer who receive (ADT) have shown increased quality of life. However, survival benefit with ADT is reduced dramatically in castration-resistant prostate cancer (CRPC). Androgen receptor (AR) continues to be functional in CRPC through various mechanisms. It is becoming evident that AR-V7, an AR variant with constitutive transcriptional activity, plays crucial roles in the development of CRPC. In order to study the mechanism of AR-V7’s actions and the ability of resveratrol to repress AR-V7, we ectopically expressed AR-V7 in PC3 cells and treated the cells with different concentrations of resveratrol. Resveratrol represses AR-V7 transcriptional activity in a dose-dependent manner evidenced by levels of the AR target gene PSA. Mechanistically, we found that under our specific experimental conditions resveratrol down-regulates AR-V7 protein levels post-transcriptionally without affecting AR-V7 subcellular location. Given the fact that resveratrol potentially represses endogenous AR-V7 transcriptional activity at the transcriptional level, resveratrol could become an option in CPRC treatment

A pharmacological cocktail for arresting actin dynamics in living cells.

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts

May the Best Molecule Win: Competition ESI Mass Spectrometry

Electrospray ionization mass spectrometry has become invaluable in the characterization of macromolecular biological systems such as nucleic acids and proteins. Recent advances in the field of mass spectrometry and the soft conditions characteristic of electrospray ionization allow for the investigation of non-covalent interactions among large biomolecules and ligands. Modulation of genetic processes through the use of small molecule inhibitors with the DNA minor groove is gaining attention as a potential therapeutic approach. In this review, we discuss the development of a competition method using electrospray ionization mass spectrometry to probe the interactions of multiple DNA sequences with libraries of minor groove binding molecules. Such an approach acts as a high-throughput screening method to determine important information including the stoichiometry, binding mode, cooperativity, and relative binding affinity. In addition to small molecule-DNA complexes, we highlight other applications in which competition mass spectrometry has been used. A competitive approach to simultaneously investigate complex interactions promises to be a powerful tool in the discovery of small molecule inhibitors with high specificity and for specific, important DNA sequences

A framework to support human factors of automation in railway intelligent infrastructure

Technological and organisational advances have increased the potential for remote access and proactive monitoring of the infrastructure in various domains and sectors – water and sewage, oil and gas and transport. Intelligent Infrastructure (II) is an architecture that potentially enables the generation of timely and relevant information about the state of any type of infrastructure asset, providing a basis for reliable decision-making. This paper reports an exploratory study to understand the concepts and human factors associated with II in the railway, largely drawing from structured interviews with key industry decision-makers and attachment to pilot projects. Outputs from the study include a data-processing framework defining the key human factors at different levels of the data structure within a railway II system and a system-level representation. The framework and other study findings will form a basis for human factors contributions to systems design elements such as information interfaces and role specifications

On the scale-height of the molecular gas disc in Milky Way-like galaxies

We study the relationship between the scale-height of the molecular gas disc and the turbulent velocity dispersion of the molecular interstellar medium within a simulation of a Milky Way-like galaxy in the moving-mesh code Arepo. We find that the vertical distribution of molecular gas can be described by a Gaussian function with a uniform scale-height of ~50 pc. We investigate whether this scale-height is consistent with a state of hydrostatic balance between gravity and turbulent pressure. We find that the hydrostatic prediction using the total turbulent velocity dispersion (as one would measure from kpc-scale observations) gives an over-estimate of the true molecular disc scale-height. The hydrostatic prediction using the velocity dispersion between the centroids of discrete giant molecular clouds (cloud-cloud velocity dispersion) leads to more-accurate estimates. The velocity dispersion internal to molecular clouds is elevated by the locally-enhanced gravitational field. Our results suggest that observations of molecular gas need to reach the scale of individual molecular clouds in order to accurately determine the molecular disc scale-height.Comment: MNRAS accepted, comments welcome. 14 pages, 10 figure

A Course-Based Research Experience: How Benefits Change with Increased Investment in Instructional Time

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit

A Novel Approach Using Electrospray Ionization Mass Spectrometry to Study Competitive Binding of Small Molecules with Mixed DNA Sequences

Minor groove binding compounds have been shown to induce changes in global DNA conformation, allosterically inhibiting DNA-protein interactions necessary for transcriptional processes. Many minor groove binders are specific for AT-base pairs but have little preference over alternating AT or A-tract sequences. Few compounds, other than polyamides, show selectivity for mixed sequences with AT and GC base pairs. Electrospray ionization mass spectrometry (ESI-MS) can provide insight on the stoichiometry and relative affinities in minor groove recognition of different DNA sequences with a library of minor groove binders. A goal in our current research is to develop new compounds that recognize mixed sequences of DNA. In an effort to optimize screening for compounds that target mixed AT and GC base pair sequences of DNA, ESI-MS was used to study the competitive binding of compounds with a mixed set of DNA sequences. The method identified preferred binding sites, relative affinities, and concentration-dependent binding stoichiometry for the minor groove binding compounds netropsin and DB75 with AT-rich sequences, and DB293 with ATGA and AT-sites