Mechanisms of Alpha-Synuclein Seeded Aggregation in Neurons Revealed by Fluorescence Lifetime Imaging.

1The brains of Parkinson's disease (PD) patients are characterized by the presence of Lewy body inclusions enriched with fibrillar forms of the presynaptic protein alpha-synuclein (aSyn). Despite related evidence that Lewy pathology spreads across different brain regions as the disease progresses, the underlying mechanism hence the fundamental cause of PD progression is unknown. The propagation of aSyn pathology is thought to potentially occur through the release of aSyn aggregates from diseased neurons, their uptake by neighboring healthy neurons via endocytosis, and subsequent seeding of native aSyn aggregation in the cytosol.

Development of an integrated sample amplification control for salivary point-of-care pathogen testing.

Background: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked.

Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing.

Background: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked.

Optimization of a Pyrimidinone Series for Selective Inhibition of Ca/Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain.

Adenylyl cyclase type 1 (AC1) is involved in signaling for chronic pain sensitization in the central nervous system and is an emerging target for the treatment of chronic pain. AC1 and a closely related isoform AC8 are also implicated to have roles in learning and memory signaling processes. Our team has carried out cellular screening for inhibitors of AC1 yielding a pyrazolyl-pyrimidinone scaffold with low micromolar potency against AC1 and selectivity AC8.

Genome-Wide Small Interfering RNA Screening Reveals a Role for Cullin3-Really Interesting New Gene Ligase Signaling in Heterologous Sensitization of Adenylyl Cyclase.

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of G -coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown.

Microfluidic rapid and autonomous analytical device (microRAAD) to detect HIV from whole blood samples.

While identifying acute HIV infection is critical to providing prompt treatment to HIV-positive individuals and preventing transmission, existing laboratory-based testing methods are too complex to perform at the point of care. Specifically, molecular techniques can detect HIV RNA within 8-10 days of transmission but require laboratory infrastructure for cold-chain reagent storage and extensive sample preparation performed by trained personnel. Here, we demonstrate our point-of-care microfluidic rapid and autonomous analysis device (microRAAD) that automatically detects HIV RNA from whole blood.

Protein Labeling and Bioconjugation Using N-Myristoyltransferase.

Methods that allow for labeling of proteins cotranslationally within protein expression systems have had wide-ranging applications in health, engineering, and medicine. Bioorthogonal chemistries that allow for conjugation of proteins or biomolecules of interest to substrates (fluorophores, gold nanoparticles, polymers, etc.) in living cells without prior enrichment or purification have likewise enabled advances in technology to study and engineer cellular and biomolecular systems.

Angiotensin II Infusion Does Not Cause Abdominal Aortic Aneurysms in Apolipoprotein E-Deficient Rats.

The apolipoprotein E-deficient (apoE-/-) mouse model has advanced our understanding of cardiovascular disease mechanisms and experimental therapeutics. This spontaneous model recapitulates aspects of human atherosclerosis, and allows for the development of dissecting abdominal aortic aneurysms (AAAs) when combined with angiotensin II. We characterized apoE-/- rats and hypothesized that, similar to mice, they would develop dissecting AAAs.

Fluorescent imaging of protein myristoylation during cellular differentiation and development.

Protein post-translational modifications (PTMs) serve to give proteins new cellular functions and can influence spatial distribution and enzymatic activity, greatly enriching the complexity of the proteome. Lipidation is a PTM that regulates protein stability, function, and subcellular localization. To complement advances in proteomic identification of lipidated proteins, we have developed a method to image the spatial distribution of proteins that have been co- and post-translationally modified via the addition of myristic acid (Myr) to the N terminus.

Comparative pharmacological characterization of D1-like dopamine receptors from Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus suggests pleiotropic signaling in mosquito vector lineages.

Background: Small molecule antagonists of mosquito dopamine receptors (DARs) are under investigation as a new class of vector-selective insecticides. Antagonists that inhibit the D1-like DARs AaDOP2 and CqDOP2 from the mosquitoes Aedes aegypti L. and Culex quinquefasciatus Say, respectively, also cause larval mortality in bioassays.

Dopamine receptor antagonists as new mode-of-action insecticide leads for control of Aedes and Culex mosquito vectors.

Background: New mode-of-action insecticides are sought to provide continued control of pesticide resistant arthropod vectors of neglected tropical diseases (NTDs). We previously identified antagonists of the AaDOP2 D1-like dopamine receptor (DAR) from the yellow fever mosquito, Aedes aegypti, with toxicity to Ae. aegypti larvae as leads for novel insecticides.

Bimolecular fluorescence complementation analysis of G protein-coupled receptor dimerization in living cells.

Emerging evidence indicates that G protein-coupled receptor (GPCR) signaling is mediated by receptor-receptor interactions at multiple levels. Thus, understanding the biochemistry and pharmacology of those receptor complexes is an important part of delineating the fundamental processes associated with GPCR-mediated signaling in human disease. A variety of experimental approaches have been used to explore these complexes, including bimolecular fluorescence complementation (BiFC) and multicolor BiFC (mBiFC).

Discovery of antagonists of tick dopamine receptors via chemical library screening and comparative pharmacological analyses.

Ticks transmit a wide variety of disease causing pathogens to humans and animals. Considering the global health impact of tick-borne diseases, there is a pressing need to develop new methods for vector control. We are exploring arthropod dopamine receptors as novel targets for insecticide/acaricide development because of their integral roles in neurobiology.

Dopamine D(2) Receptor-Mediated Heterologous Sensitization of AC5 Requires Signalosome Assembly.

Chronic dopamine receptor activation is implicated in several central nervous system disorders. Although acute activation of Gα(i)-coupled D(2) dopamine receptors inhibits adenylyl cyclase, persistent activation enhances adenylyl cyclase activity, a phenomenon called heterologous sensitization. Previous work revealed a requirement for Gα(s) in D(2)-induced heterologous sensitization of AC5.

A "genome-to-lead" approach for insecticide discovery: pharmacological characterization and screening of Aedes aegypti D(1)-like dopamine receptors.

Background: Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides.

Methodology/principal Findings: We describe a "genome-to-lead" approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome.

Molecular and pharmacological characterization of two D(1)-like dopamine receptors in the Lyme disease vector, Ixodes scapularis.

Advancements in tick neurobiology may impact the development of acaricides to control those species that transmit human and animal diseases. Here, we report the first cloning and pharmacological characterization of two neurotransmitter binding G protein-coupled receptors in the Lyme disease (blacklegged) tick, Ixodes scapularis. The genes IscaGPRdop1 and IscaGPRdop2 were identified in the I.

Human cord blood-derived mast cells are activated by the Nod1 agonist M-TriDAP to release pro-inflammatory cytokines and chemokines.

Mast cells are among the first cells of our immune system to encounter exogenous danger. Intracellular receptors such as nucleotide-binding oligomerization domain (Nod) play an important role in responding to invading pathogens. Here, we have investigated the response of human mast cells to the Nod1 ligand M-TriDAP.

Fluorescent protein complementation assays: new tools to study G protein-coupled receptor oligomerization and GPCR-mediated signaling.

G protein-coupled receptor (GPCR) signaling is mediated by protein-protein interactions at multiple levels. The characterization of the corresponding protein complexes is therefore paramount to the basic understanding of GPCR-mediated signal transduction. The number of documented interactions involving GPCRs is rapidly growing, and appreciating the functional significance of these complexes is clearly the next challenge.

Substrate overlap between Mrp4 and Abcg2/Bcrp affects purine analogue drug cytotoxicity and tissue distribution.

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA.

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding.

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells.

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1.

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1.

Differential sensitivities of the human ATP-binding cassette transporters ABCG2 and P-glycoprotein to cyclosporin A.

Several ATP-binding cassette (ABC) transporters can confer multidrug resistance to cancer cells by functioning as energy-dependent efflux pumps. The half-transporter ABCG2 and the widely studied P-glycoprotein (P-gp) are two ABC transporters that, when overexpressed, are capable of extruding a variety of structurally unrelated chemotherapy agents from cells. In this study, we demonstrate that human ABCG2 and P-glycoprotein, despite overlapping substrate specificities, differ in sensitivity to the immunomodulator cyclosporin A.

Multidrug resistance and cancer: the role of the human ABC transporter ABCG2.

A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional chemotherapy, a phenomenon called multidrug resistance. This broad-based resistance results in large part, but not solely, from overexpression of members of the ATP-binding cassette (ABC) superfamily of membrane transporters, including P-glycoprotein, various members of the multidrug resistance associated proteins (MRPs), and ABCG2, also known as MXR1, BCRP, and ABCP. When overexpressed in cell lines, ABCG2 has the ability to confer high levels of resistance to anthracyclines, mitoxantrone, bisantrene, and the camptothecins topotecan and SN-38.