Targeting the Poly (ADP-Ribose) Polymerase-1 Catalytic Pocket Using AutoGrow4, a Genetic Algorithm for De Novo Design

AutoGrow4 is a free and open-source program for de novo drug design that uses a genetic algorithm (GA) to create novel predicted small-molecule ligands for a given protein target without the constraints of a finite, pre-defined virtual library. By leveraging recent computational and cheminformatic advancements, AutoGrow4 is faster, more stable, and more modular than previous versions. Features such as docking-software compatibility, chemical filters, multithreading options, and selection methods have been expanded to support a wide range of user needs. This dissertation will cover the development and validation of AutoGrow4, as well as its application to poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a well-characterized DNA-damage recognition protein, and PARP-1 inhibition is an effective treatment for ovarian and breast cancers that are homologous-recombination (HR) deficient1–5. As a well-studied protein, PARP-1 is also an excellent drug target with which to validate AutoGrow4. Multiple crystallographic structures of PARP-1 bound to various PARP-1 inhibitors (PARPi) serve as positive controls for assessing the quality of AutoGrow4-generated compounds in terms of predicted binding affinity, chemical structure, and predicted protein-ligand interactions. This dissertation describes how I (1) generated novel potential PARPi with predicted binding affinities that surpass those of known PARPi; (2) validated AutoGrow4 as a tool for de novo drug design, lead optimization, and hypothesis generation, using PARP-1 as a test target; (3) contributed support to the growing notion that there is a need for HR-deficient cancer chemotherapies that do not rely on the same set of protein-ligand interactions typical of current PARPi; (4) generated novel potential PARPi that are predicted to bind to PARP-1 independent of a post-translational modification that is known to cause PARPi resistance; and (5) generated novel potential PARPi that are predicted to bind a secondary PARP-1 pocket that is distant from the primary catalytic site

Gypsum-DL: an open-source program for preparing small-molecule libraries for structure-based virtual screening

Computational techniques such as structure-based virtual screening require carefully prepared 3D models of potential small-molecule ligands. Though powerful, existing commercial programs for virtual-library preparation have restrictive and/or expensive licenses. Freely available alternatives, though often effective, do not fully account for all possible ionization, tautomeric, and ring-conformational variants. We here present Gypsum-DL, a free, robust open-source program that addresses these challenges. As input, Gypsum-DL accepts virtual compound libraries in SMILES or flat SDF formats. For each molecule in the virtual library, it enumerates appropriate ionization, tautomeric, chiral, cis/trans isomeric, and ring-conformational forms. As output, Gypsum-DL produces an SDF file containing each molecular form, with 3D coordinates assigned. To demonstrate its utility, we processed 1558 molecules taken from the NCI Diversity Set VI and 56,608 molecules taken from a Distributed Drug Discovery (D3) combinatorial virtual library. We also used 4463 high-quality protein-ligand complexes from the PDBBind database to show that Gypsum-DL processing can improve virtual-screening pose prediction. Gypsum-DL is available free of charge under the terms of the Apache License, Version 2.0

Considerations for preparing a randomized population health intervention trial: lessons from a South African–Canadian partnership to improve the health of health workers

Background: Community-based cluster-randomized controlled trials (RCTs) are increasingly being conducted to address pressing global health concerns. Preparations for clinical trials are well-described, as are the steps for multi-component health service trials. However, guidance is lacking for addressing the ethical and logistic challenges in (cluster) RCTs of population health interventions in low- and middle-income countries. Objective: We aimed to identify the factors that population health researchers must explicitly consider when planning RCTs within North–South partnerships. Design: We reviewed our experiences and identified key ethical and logistic issues encountered during the pre-trial phase of a recently implemented RCT. This trial aimed to improve tuberculosis (TB) and Human Immunodeficiency Virus (HIV) prevention and care for health workers by enhancing workplace assessment capability, addressing concerns about confidentiality and stigma, and providing onsite counseling, testing, and treatment. An iterative framework was used to synthesize this analysis with lessons taken from other studies. Results: The checklist of critical factors was grouped into eight categories: 1) Building trust and shared ownership; 2) Conducting feasibility studies throughout the process; 3) Building capacity; 4) Creating an appropriate information system; 5) Conducting pilot studies; 6) Securing stakeholder support, with a view to scale-up; 7) Continuously refining methodological rigor; and 8) Explicitly addressing all ethical issues both at the start and continuously as they arise. Conclusion: Researchers should allow for the significant investment of time and resources required for successful implementation of population health RCTs within North–South collaborations, recognize the iterative nature of the process, and be prepared to revise protocols as challenges emerge

Flexor Hallucis Longus tendon rupture in RA-patients is associated with MTP 1 damage and pes planus

<p>Abstract</p> <p>Background</p> <p>To assess the prevalence of and relation between rupture or tenosynovitis of the Flexor Hallucis Longus (FHL) tendon and range of motion, deformities and joint damage of the forefoot in RA patients with foot complaints.</p> <p>Methods</p> <p>Thirty RA patients with painful feet were analysed, their feet were examined clinically for the presence of pes planus and range of motion (ROM), radiographs were scored looking for the presence of forefoot damage, and ultrasound examination was performed, examining the presence of tenosyovitis or rupture of the FHL at the level of the medial malleolus. The correlation between the presence or absence of the FHL and ROM, forefoot damage and pes planus was calculated.</p> <p>Results</p> <p>In 11/60(18%) of the feet, a rupture of the FHL was found. This was associated with a limited motion of the MTP1-joint, measured on the JAM (χ²= 10.4, p = 0.034), a higher prevalence of pes planus (χ²= 5.77, p = 0.016) and a higher prevalence of erosions proximal at the MTP-1 joint (χ²= 12.3, p = 0.016), and joint space narrowing of the MTP1 joint (χ²= 12.7, p = 0.013).</p> <p>Conclusion</p> <p>Rupture of the flexor hallucis longus tendon in RA-patients is associated with limited range of hallux motion, more erosions and joint space narrowing of the MTP-1-joint, as well as with pes planus.</p

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signalling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signalling molecule is sphingosine-1-phosphate (S1P) whose breakdown is catalysed by sphingosine-1-phosphate lyase (S1PL), a pyridoxal 5 '-phosphate (PLP) dependent enzyme that catalyses the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine (PE). Here we show the pathogenic bacterium Burkholderia pseudomallei K96243 encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established mass spectrometry-based methodology we show that recombinant S1PL2021 is catalytically active. Using recombinant human fatty aldehyde dehydrogenase (FALDH) we developed a spectrophotometric, enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the x-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.The authors thanks the following for funding: The Biotechnology and Biological Sciences Research Council (BBSRC) for an EastBio Doctoral Training Programme PhD studentship award to C McLean (BB/J01446X/1) and a grant awarded to DJ Campopiano (BB/I013687/1) that supported J Lowther and DJ Clarke. R Custodio was supported by the Defence Science and Technology Laboratory under contract DSTLX-1000060221 (WP1). We thank the staff of the Diamond Light Source, UK for help with data collection. The authors thank Prof. John RW Govan (University of Edinburgh) for his suggestions regarding Burkholderia strains and enthusiastic support of this work. We also thanks Dr. Kevin Ralston for help in the synthesis of 2E-HEX. The data associated with this paper is available to download (http://dx.doi.org/10.7488/ds/1412)

A De Novo Protein Binding Pair By Computational Design and Directed Evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity- matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180 relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demon- strates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and under- scores remaining challenges.Chemistry and Chemical Biolog

The nuclear configuration and cortical connections of the human thalamus

No Abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49943/1/900830102_ftp.pd

A comprehensive analysis of autocorrelation and bias in home range estimation

Home range estimation is routine practice in ecological research. While advances in animal tracking technology have increased our capacity to collect data to support home range analysis, these same advances have also resulted in increasingly autocorrelated data. Consequently, the question of which home range estimator to use on modern, highly autocorrelated tracking data remains open. This question is particularly relevant given that most estimators assume independently sampled data. Here, we provide a comprehensive evaluation of the effects of autocorrelation on home range estimation. We base our study on an extensive data set of GPS locations from 369 individuals representing 27 species distributed across five continents. We first assemble a broad array of home range estimators, including Kernel Density Estimation (KDE) with four bandwidth optimizers (Gaussian reference function, autocorrelated-Gaussian reference function [AKDE], Silverman´s rule of thumb, and least squares cross-validation), Minimum Convex Polygon, and Local Convex Hull methods. Notably, all of these estimators except AKDE assume independent and identically distributed (IID) data. We then employ half-sample cross-validation to objectively quantify estimator performance, and the recently introduced effective sample size for home range area estimation ((Formula presented.)) to quantify the information content of each data set. We found that AKDE 95% area estimates were larger than conventional IID-based estimates by a mean factor of 2. The median number of cross-validated locations included in the hold-out sets by AKDE 95% (or 50%) estimates was 95.3% (or 50.1%), confirming the larger AKDE ranges were appropriately selective at the specified quantile. Conversely, conventional estimates exhibited negative bias that increased with decreasing (Formula presented.). To contextualize our empirical results, we performed a detailed simulation study to tease apart how sampling frequency, sampling duration, and the focal animal´s movement conspire to affect range estimates. Paralleling our empirical results, the simulation study demonstrated that AKDE was generally more accurate than conventional methods, particularly for small (Formula presented.). While 72% of the 369 empirical data sets had >1,000 total observations, only 4% had an (Formula presented.) >1,000, where 30% had an (Formula presented.) <30. In this frequently encountered scenario of small (Formula presented.), AKDE was the only estimator capable of producing an accurate home range estimate on autocorrelated data.Fil: Noonan, Michael J.. National Zoological Park; Estados Unidos. University of Maryland; Estados UnidosFil: Tucker, Marlee A.. Senckenberg Gesellschaft Für Naturforschung; . Goethe Universitat Frankfurt; AlemaniaFil: Fleming, Christen H.. University of Maryland; Estados Unidos. National Zoological Park; Estados UnidosFil: Akre, Thomas S.. National Zoological Park; Estados UnidosFil: Alberts, Susan C.. University of Duke; Estados UnidosFil: Ali, Abdullahi H.. Hirola Conservation Programme. Garissa; KeniaFil: Altmann, Jeanne. University of Princeton; Estados UnidosFil: Antunes, Pamela Castro. Universidade Federal do Mato Grosso do Sul; BrasilFil: Belant, Jerrold L.. State University of New York; Estados UnidosFil: Beyer, Dean. Universitat Phillips; AlemaniaFil: Blaum, Niels. Universitat Potsdam; AlemaniaFil: Böhning Gaese, Katrin. Senckenberg Gesellschaft Für Naturforschung; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Cullen Jr., Laury. Instituto de Pesquisas Ecológicas; BrasilFil: de Paula, Rogerio Cunha. National Research Center For Carnivores Conservation; BrasilFil: Dekker, Jasja. Jasja Dekker Dierecologie; Países BajosFil: Drescher Lehman, Jonathan. George Mason University; Estados Unidos. National Zoological Park; Estados UnidosFil: Farwig, Nina. Michigan State University; Estados UnidosFil: Fichtel, Claudia. German Primate Center; AlemaniaFil: Fischer, Christina. Universitat Technical Zu Munich; AlemaniaFil: Ford, Adam T.. University of British Columbia; CanadáFil: Goheen, Jacob R.. University of Wyoming; Estados UnidosFil: Janssen, René. Bionet Natuuronderzoek; Países BajosFil: Jeltsch, Florian. Universitat Potsdam; AlemaniaFil: Kauffman, Matthew. University Of Wyoming; Estados UnidosFil: Kappeler, Peter M.. German Primate Center; AlemaniaFil: Koch, Flávia. German Primate Center; AlemaniaFil: LaPoint, Scott. Max Planck Institute für Ornithologie; Alemania. Columbia University; Estados UnidosFil: Markham, A. Catherine. Stony Brook University; Estados UnidosFil: Medici, Emilia Patricia. Instituto de Pesquisas Ecológicas (IPE) ; BrasilFil: Morato, Ronaldo G.. Institute For Conservation of The Neotropical Carnivores; Brasil. National Research Center For Carnivores Conservation; BrasilFil: Nathan, Ran. The Hebrew University of Jerusalem; IsraelFil: Oliveira Santos, Luiz Gustavo R.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Olson, Kirk A.. Wildlife Conservation Society; Estados Unidos. National Zoological Park; Estados UnidosFil: Patterson, Bruce. Field Museum of National History; Estados UnidosFil: Paviolo, Agustin Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Ramalho, Emiliano Esterci. Institute For Conservation of The Neotropical Carnivores; Brasil. Instituto de Desenvolvimento Sustentavel Mamirauá; BrasilFil: Rösner, Sascha. Michigan State University; Estados UnidosFil: Schabo, Dana G.. Michigan State University; Estados UnidosFil: Selva, Nuria. Institute of Nature Conservation of The Polish Academy of Sciences; PoloniaFil: Sergiel, Agnieszka. Institute of Nature Conservation of The Polish Academy of Sciences; PoloniaFil: Xavier da Silva, Marina. Parque Nacional do Iguaçu; BrasilFil: Spiegel, Orr. Universitat Tel Aviv; IsraelFil: Thompson, Peter. University of Maryland; Estados UnidosFil: Ullmann, Wiebke. Universitat Potsdam; AlemaniaFil: Ziḝba, Filip. Tatra National Park; PoloniaFil: Zwijacz Kozica, Tomasz. Tatra National Park; PoloniaFil: Fagan, William F.. University of Maryland; Estados UnidosFil: Mueller, Thomas. Senckenberg Gesellschaft Für Naturforschung; . Goethe Universitat Frankfurt; AlemaniaFil: Calabrese, Justin M.. National Zoological Park; Estados Unidos. University of Maryland; Estados Unido

Strong HIV-1-Specific T Cell Responses in HIV-1-Exposed Uninfected Infants and Neonates Revealed after Regulatory T Cell Removal

BACKGROUND: In utero transmission of HIV-1 occurs on average in only 3%–15% of HIV-1-exposed neonates born to mothers not on antiretroviral drug therapy. Thus, despite potential exposure, the majority of infants remain uninfected. Weak HIV-1-specific T-cell responses have been detected in children exposed to HIV-1, and potentially contribute to protection against infection. We, and others, have recently shown that the removal of CD4(+)CD25(+) T-regulatory (Treg) cells can reveal strong HIV-1 specific T-cell responses in some HIV-1 infected adults. Here, we hypothesized that Treg cells could suppress HIV-1-specific immune responses in young children. METHODOLOGY/PRINCIPAL FINDINGS: We studied two cohorts of children. The first group included HIV-1-exposed-uninfected (EU) as well as unexposed (UNEX) neonates. The second group comprised HIV-1-infected and HIV-1-EU children. We quantified the frequency of Treg cells, T-cell activation, and cell-mediated immune responses. We detected high levels of CD4(+)CD25(+)CD127(−) Treg cells and low levels of CD4(+) and CD8(+) T cell activation in the cord blood of the EU neonates. We observed HIV-1-specific T cell immune responses in all of the children exposed to the virus. These T-cell responses were not seen in the cord blood of control HIV-1 unexposed neonates. Moreover, the depletion of CD4(+)CD25(+) Treg cells from the cord blood of EU newborns strikingly augmented both CD4(+) and CD8(+) HIV-1-specific immune responses. CONCLUSIONS/SIGNIFICANCE: This study provides new evidence that EU infants can mount strong HIV-1-specific T cell responses, and that in utero CD4(+)CD25(+) T-regulatory cells may be contributing to the lack of vertical transmission by reducing T cell activation