@article {166, title = {Dual-target, real-time PCR for the diagnosis of intraocular infections.}, journal = {Br J Ophthalmol}, volume = {103}, year = {2019}, month = {2019 04}, pages = {569-572}, abstract = {

BACKGROUND: is the most common infectious cause of posterior uveitis worldwide. Two multicopy targets (B1 and Rep529) are commonly used in PCR assays, but studies evaluating these targets in ocular fluid samples are limited. Herein, we determine the analytical characteristics of a single-reaction, internally controlled, dual-target, real-time PCR and evaluate the clinical performance of this assay in intraocular fluid samples obtained at a reference ophthalmologic centre in the USA.

METHODS: Lower limits of detection for the B1 and Rep529 components of the dual-target assay were determined using serial dilutions of cultured strain Z185. The dual-target assay was then used to test 148 archived intraocular samples (132 vitreous,16 aqueous humour) collected at the Francis I. Proctor Foundation between January 2010 and December 2015 for testing by a nested, conventional PCR targeting the B1 gene.

RESULTS: The 95\% lower limits of detection for the dual-target assay was determined to be 1.05 tachyzoites/mL for B1 and 0.83 tachyzoites/mL for Rep529. Using archived clinical intraocular specimens, the dual-target assay demonstrated 97.2\% positive per cent agreement (n=35/36; 95\% CI 83.7\% to 99.9\%) and 99.1\% negative per cent agreement (n=111/112; 95\% CI 94.4\% to 100\%) compared with the nested, conventional B1 PCR.

CONCLUSION: This single-reaction, internally controlled, dual-target (B1, Rep529) real-time PCR for the detection of DNA in intraocular specimens demonstrated excellent agreement with nested, conventional, B1 PCR. The dual-target design may ensure detection when variation is present in one of two target regions.

}, keywords = {Animals, Aqueous Humor, DNA, Protozoan, Eye Infections, Parasitic, Humans, Real-Time Polymerase Chain Reaction, Toxoplasma, Toxoplasmosis, Ocular, Uveitis, Posterior}, issn = {1468-2079}, doi = {10.1136/bjophthalmol-2018-313064}, author = {Gomez, Carlos A and Sahoo, Malaya K and Kahn, Ghazala Yasmeen and Zhong, Lina and Montoya, Jos{\'e} G and Pinsky, Benjamin A and Doan, Thuy} } @article {186, title = {Metagenomic Deep Sequencing for the Diagnosis of Corneal and External Disease Infections.}, journal = {Ophthalmology}, volume = {126}, year = {2019}, month = {2019 12}, pages = {1724-1726}, keywords = {Adult, Aged, Aged, 80 and over, Animals, Bacteria, Conjunctival Diseases, Corneal Diseases, Eye Infections, Female, Fungi, High-Throughput Nucleotide Sequencing, Humans, Male, Metagenome, Middle Aged, Parasites, Polymerase Chain Reaction, Retrospective Studies, Scleral Diseases}, issn = {1549-4713}, doi = {10.1016/j.ophtha.2019.06.013}, author = {Seitzman, Gerami D and Hinterwirth, Armin and Zhong, Lina and Cummings, Susie and Chen, Cindi and Driver, Todd H and Lee, Michele D and Doan, Thuy} } @article {291, title = {C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor.}, journal = {Elife}, volume = {4}, year = {2015}, month = {2015 Apr 24}, abstract = {

Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.

}, keywords = {Animals, Arrestin, Binding Sites, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphorylation, Receptors, G-Protein-Coupled, Retinal Rod Photoreceptor Cells, Rhodopsin, Serine, Threonine}, issn = {2050-084X}, doi = {10.7554/eLife.05981}, author = {Azevedo, Anthony W and Doan, Thuy and Moaven, Hormoz and Sokal, Iza and Baameur, Faiza and Vishnivetskiy, Sergey A and Homan, Kristoff T and Tesmer, John J G and Gurevich, Vsevolod V and Chen, Jeannie and Rieke, Fred} } @article {741, title = {Arrestin competition influences the kinetics and variability of the single-photon responses of mammalian rod photoreceptors.}, journal = {J Neurosci}, volume = {29}, year = {2009}, month = {2009 Sep 23}, pages = {11867-79}, abstract = {

Reliable signal transduction via G-protein-coupled receptors requires proper receptor inactivation. For example, signals originating from single rhodopsin molecules vary little from one to the next, requiring reproducible inactivation of rhodopsin by phosphorylation and arrestin binding. We determined how reduced concentrations of rhodopsin kinase (GRK1) and/or arrestin1 influenced the kinetics and variability of the single-photon responses of mouse rod photoreceptors. These experiments revealed that arrestin, in addition to its role in quenching the activity of rhodopsin, can tune the kinetics of rhodopsin phosphorylation by competing with GRK1. This competition influenced the variability of the active lifetime of rhodopsin. Biasing the competition in favor of GRK1 revealed that rhodopsin remained active through much of the single-photon response under the conditions of our experiments. This long-lasting rhodopsin activity can explain the characteristic time course of single-photon response variability. Indeed, explaining the late time-to-peak of the variance required an active lifetime of rhodopsin approximately twice that of the G-protein transducin. Competition between arrestins and kinases may be a general means of influencing signals mediated by G-protein-coupled receptors, particularly when activation of a few receptors produces signals of functional importance.

}, keywords = {Animals, Arrestin, G-Protein-Coupled Receptor Kinase 1, Kinetics, Markov Chains, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Neurological, Phosphorylation, Photic Stimulation, Photons, Retinal Rod Photoreceptor Cells, Rhodopsin, Signal Transduction, Time Factors}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0819-09.2009}, author = {Doan, Thuy and Azevedo, Anthony W and Hurley, James B and Rieke, Fred} } @article {726, title = {Multiple phosphorylation sites confer reproducibility of the rod{\textquoteright}s single-photon responses.}, journal = {Science}, volume = {313}, year = {2006}, month = {2006 Jul 28}, pages = {530-3}, abstract = {

Although signals controlled by single molecules are expected to be inherently variable, rod photoreceptors generate reproducible responses to single absorbed photons. We show that this unexpected reproducibility-the consistency of amplitude and duration of rhodopsin activity-varies in a graded and systematic manner with the number but not the identity of phosphorylation sites on rhodopsin{\textquoteright}s C terminus. These results indicate that each phosphorylation site provides an independent step in rhodopsin deactivation and that collectively these steps tightly control rhodopsin{\textquoteright}s active lifetime. Other G protein cascades may exploit a similar mechanism to encode accurately the timing and number of receptor activation.

}, keywords = {Animals, Arrestin, Electrophysiology, Mice, Mice, Transgenic, Models, Biological, Mutation, Patch-Clamp Techniques, Phosphorylation, Photons, Retinal Rod Photoreceptor Cells, Rhodopsin, Vision, Ocular}, issn = {1095-9203}, doi = {10.1126/science.1126612}, author = {Doan, Thuy and Mendez, Ana and Detwiler, Peter B and Chen, Jeannie and Rieke, Fred} }