IDENTIFICATION OF NOVEL POTENTIAL CANCER THERAPIES BY SYNTHETIC LETHAL SCREENING

There is an urgent need for novel effective drug regimens for the treatment of cancer. Current chemotherapy suffers from a slim therapeutic index, with significant toxicity from effective drug doses or tumor recurrence at low drug doses. Identifying synergistic interactions between drugs is a difficult process. To accelerate the discovery of potential drug combinations, I have developed a druggable genome siRNA synthetic lethal screen capable of rapidly identifying novel drug targets that would sensitize cancer cells to sublethal concentrations of microtubule destabilizing agents. I employed a high-throughput cell-based 16,560-siRNA screen to isolate a high-confidence list of genes that, when silenced, enhanced glioblastoma multiforme cancer cell chemosensitivity. Two gene products that were the major focus of my work were midline2 and the neurokinin receptor NK1R. Silencing of midline2, a PP2A-microtubule tether, sensitized cells to two microtubule destabilizing agents, vinblastine and disorazole C1, suggesting a mechanistic dependency of the phosphatidylinositol 3-kinase pathway on microtubule functionality. Combinations of phosphatidylinositol 3-kinase inhibitors with disorazole C1 and several vinca alkaloids confirmed this hypothesis. To verify microtubule destabilizing agent sensitization by NK1R silencing, I demonstrated a significant collaboration of neurokinin receptor NK1R antagonists with low concentrations of vinca alkaloids. These assay results and subsequent novel combination strategies demonstrate the tremendous ability of this synthetic lethal screen to predict potent collaborations between different classes of drugs, as well as identifying molecular constituents mediating those interactions

IDCS J1426.5+3508: The Most Massive Galaxy Cluster at z>1.5z > 1.5

We present a deep (100 ks) Chandra observation of IDCS J1426.5+3508, a spectroscopically confirmed, infrared-selected galaxy cluster at $z = 1.75$. This cluster is the most massive galaxy cluster currently known at $z > 1.5$, based on existing Sunyaev-Zel'dovich (SZ) and gravitational lensing detections. We confirm this high mass via a variety of X-ray scaling relations, including $T_X$-M, $f_g$-M, $Y_X$-M and $L_X$-M, finding a tight distribution of masses from these different methods, spanning M$_{500}$ = 2.3-3.3 $\times 10^{14}$ M$_{\odot}$, with the low-scatter $Y_X$-based mass $M_{500,Y_X} = 2.6^{+1.5}_{-0.5} \times 10^{14}$ M$_\odot$. IDCS J1426.5+3508 is currently the only cluster at $z > 1.5$ for which X-ray, SZ and gravitational lensing mass estimates exist, and these are in remarkably good agreement. We find a relatively tight distribution of the gas-to-total mass ratio, employing total masses from all of the aforementioned indicators, with values ranging from $f_{gas,500}$ = 0.087-0.12. We do not detect metals in the intracluster medium (ICM) of this system, placing a 2$\sigma$ upper limit of $Z(r < R_{500}) < 0.18 Z_{\odot}$. This upper limit on the metallicity suggests that this system may still be in the process of enriching its ICM. The cluster has a dense, low-entropy core, offset by $\sim$30 kpc from the X-ray centroid, which makes it one of the few "cool core" clusters discovered at $z > 1$, and the first known cool core cluster at $z > 1.2$. The offset of this core from the large-scale centroid suggests that this cluster has had a relatively recent ($\lesssim$500 Myr) merger/interaction with another massive system.Comment: Minor changes to match accepted version, results unchanged; ApJ in pres

Watershed-scale impacts of stormwater green infrastructure on hydrology, nutrient fluxes, and combined sewer overflows in the mid-Atlantic region

AbstractStormwater green infrastructure (SGI), including rain gardens, detention ponds, bioswales, and green roofs, is being implemented in cities across the globe to reduce flooding, combined sewer overflows, and pollutant transport to streams and rivers. Despite the increasing use of urban SGI, few studies have quantified the cumulative effects of multiple SGI projects on hydrology and water quality at the watershed scale. To assess the effects of SGI, Washington, DC, Montgomery County, MD, and Baltimore County, MD, were selected based on the availability of data on SGI, water quality, and stream flow. The cumulative impact of SGI was evaluated over space and time by comparing watersheds with and without SGI, and by assessing how long-term changes in SGI impact hydrologic and water quality metrics over time. Most Mid-Atlantic municipalities have a goal of achieving 10–20% of the landscape drain runoff through SGI by 2030. Of these areas, Washington, DC currently has the greatest amount of SGI (12.7% of the landscape drained through SGI), while Baltimore County has the lowest (7.9%). When controlling for watersheds size and percent impervious surface cover, watersheds with greater amounts of SGI have less flashy hydrology, with 44% lower peak runoff, 26% less frequent runoff events, and 26% less variable runoff. Watersheds with more SGI also show 44% less NO3− and 48% less total nitrogen exports compared to watersheds with minimal SGI. There was no significant reduction in phosphorus exports or combined sewer overflows in watersheds with greater SGI. When comparing individual watersheds over time, increases in SGI corresponded to non-significant reductions in hydrologic flashiness compared to watersheds with no change in SGI. While the implementation of SGI is somewhat in its infancy in some regions, cities are beginning to have a scale of SGI where there are statistically significant differences in hydrologic patterns and water quality

Open Access High Throughput Drug Discovery in the Public Domain: A Mount Everest in the Making

High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industry’s best practices for a successful outcome

The Establishment and Spread of a Newly Introduced Begomovirus in a Dry Tropical Environment Using Tomato Yellow Leaf Curl Virus as a Case Study

Early detection of tomato yellow leaf curl virus (TYLCV) in a previously unaffected tomato production district in Australia allowed its spread to be evaluated spatially and temporally. The population dynamics of the TYLCV vector, Bemisia argentifolii (silverleaf whitefly, SLW), were also evaluated. The district is a dry tropical environment with a clear break to commercial production during the summer wet season. The incidence of TYLCV within crops and its prevalence through the district was influenced by weather, location, vector movements, and the use of Ty-1 virus-resistant hybrids. Rainfall had an important influence, with late summer and early autumn rain suppressing the levels of SLW and, by contrast, a dry summer supporting faster population growth. The use of Ty-1 hybrids appears to have reduced the incidence of TYLCV in this district. There was limited use of Ty-1 hybrids during 2013, and by season end, crops had moderate levels of SLW and high virus incidence. The 2015 and early 2016 season had high SLW populations, but TYLCV incidence was lower than in 2013, possibly due to the widespread adoption of the Ty-1 hybrids reducing virus spread. This study provides valuable epidemiology data for future incursions of begomoviruses, and other viruses spread by SLW

Measuring and statistically testing the size of the effect of a chemical compound on a continuous in-vitro pharmacological response through a new statistical model of response detection limit

This is an Accepted Manuscript of an article published by Taylor & Francis in the Journal of Biopharmaceutical Statistics in June 2015, available online: http://www.tandfonline.com/10.1080/10543406.2014.920871.Biomolecular screening research frequently searches for the chemical compounds that are most likely to make a biochemical or cell-based assay system produce a strong continuous response. Several doses are tested with each compound and it is assumed that, if there is a dose-response relationship, the relationship follows a monotonic curve, usually a version of the median-effect equation. However, the null hypothesis of no relationship cannot be statistically tested using this equation. We used a linearized version of this equation to define a measure of pharmacological effect size, and use this measure to rank the investigated compounds in order of their overall capability to produce strong responses. The null hypothesis that none of the examined doses of a particular compound produced a strong response can be tested with this approach. The proposed approach is based on a new statistical model of the important concept of response detection limit, a concept that is usually neglected in the analysis of dose-response data with continuous responses. The methodology is illustrated with data from a study searching for compounds that neutralize the infection by a human immunodeficiency virus of brain glioblastoma cells

Randomised controlled feasibility trial of an evidence-informed behavioural intervention for obese adults with additional risk factors

Peer reviewedPublisher PD

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

© 2020, The Author(s). Background: Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results: A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions: We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae

YM155 Inhibits NleB and SseK Arginine Glycosyltransferase Activity

The type III secretion system effector proteins NleB and SseK are glycosyltransferases that glycosylate protein substrates on arginine residues. We conducted high-throughput screening assays on 42,498 compounds to identify NleB/SseK inhibitors. Such small molecules may be useful as mechanistic probes and may have utility in the eventual development of anti-virulence therapies against enteric bacterial pathogens. We observed that YM155 (sepantronium bromide) inhibits the activity of Escherichia coli NleB1, Citrobacter rodentium NleB, and both Salmonella enterica SseK1 and SseK2. YM155 was not toxic to mammalian cells, nor did it show cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase (OGT). YM155 reduced Salmonella survival in mouse macrophage-like cells but had no direct impact on bacterial growth rates, suggesting YM155 may have utility as a potential anti-virulence inhibitor

Repurposing Avasimibe to Inhibit Bacterial Glycosyltransferases

We are interested in identifying and characterizing small molecule inhibitors of bacterial virulence factors for their potential use as anti-virulence inhibitors. We identified from high-throughput screening assays a potential activity for avasimibe, a previously characterized acyl-coenzyme A: cholesterol acyltransferase inhibitor, in inhibiting the NleB and SseK arginine glycosyltransferases from Escherichia coli and Salmonella enterica, respectively. Avasimibe inhibited the activity of the Citrobacter rodentium NleB, E. coli NleB1, and S. enterica SseK1 enzymes, without affecting the activity of the human serine/threonine N-acetylglucosamine (O-GlcNAc) transferase. Avasimibe was not toxic to mammalian cells at up to 200 µM and was neither bacteriostatic nor bactericidal at concentrations of up to 125 µM. Doses of 10 µM avasimibe were sufficient to reduce S. enterica abundance in RAW264.7 macrophage-like cells, and intraperitoneal injection of avasimibe significantly reduced C. rodentium survival in mice, regardless of whether the avasimibe was administered pre- or post-infection. We propose that avasimibe or related derivates created using synthetic chemistry may have utility in preventing or treating bacterial infections by inhibiting arginine glycosyltransferases that are important to virulence