Thuy Doan

Controlling the gain of rod-mediated signals in the Mammalian retina.

Effective sensory processing requires matching the gain of neural responses to the range of signals encountered. For rod vision, gain controls operate at light levels at which photons arrive rarely at individual rods, light levels too low to cause adaptation in rod phototransduction. Under these conditions, adaptation within a conserved pathway in mammalian retina maintains sensitivity as light levels change. To relate retinal signals to behavioral work on detection at low light levels, we measured how background light affects the gain and noise of primate ganglion cells. To determine where and how gain is controlled, we tracked rod-mediated signals across the mouse retina. These experiments led to three main conclusions: (1) the primary site of adaptation at low light levels is the synapse between rod bipolar and AII amacrine cells; (2) cellular noise after the gain control is nearly independent of background intensity; and (3) at low backgrounds, noise in the circuitry, rather than rod noise or fluctuations in arriving photons, limits ganglion cell sensitivity. This work provides physiological insights into the rich history of experiments characterizing how rod vision avoids saturation as light levels increase.

Deletion of PrBP/δ impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments

The mouse Pde6d gene encodes a ubiquitous prenyl binding protein, termed PrBP/δ, of largely unknown physiological function. PrBP/δ was originally identified as a putative rod cGMP phosphodiesterase (PDE6) subunit in the retina, where it is relatively abundant. To investigate the consequences of Pde6d deletion in retina, we generated a Pde6d−/− mouse by targeted recombination. Although manifesting reduced body weight, the Pde6d−/− mouse was viable and fertile and its retina developed normally. Immunocytochemistry showed that farnesylated rhodopsin kinase (GRK1) and prenylated rod PDE6 catalytic subunits partially mislocalized in Pde6d−/− rods, whereas rhodopsin was unaffected. In Pde6d−/− rod single-cell recordings, sensitivity to single photons was increased and saturating flash responses were prolonged. Pde6d−/− scotopic paired-flash electroretinograms indicated a delay in recovery of the dark state, likely due to reduced levels of GRK1 in rod outer segments. In Pde6d−/− cone outer segments, GRK1 and cone PDE6α′ were present at very low levels and the photopic b-wave amplitudes were reduced by 70%. Thus the absence of PrBP/δ in retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy.

Scotopic Visual Signaling in the Mouse Retina Is Modulated by High-Affinity Plasma Membrane Calcium Extrusion

Transmission of visual signals at the first retinal synapse is associated with changes in calcium concentration in photoreceptors and bipolar cells. We investigated how loss of plasma membrane Ca2+ ATPase isoform 2 (PMCA2), the calcium transporter isoform with the highest affinity for Ca2+/calmodulin, affects transmission of rod- and cone-mediated responses. PMCA2 expression in the neuroblast layer was observed soon after birth; in the adult, PMCA2 was expressed in inner segments and synaptic terminals of rod photoreceptors, in rod bipolar cells, and in most inner retinal neurons but was absent from cones. To determine the role of PMCA2 in retinal signaling, we compared morphology and light responses of retinas from control mice and deafwaddler dfw2J mice, which lack functional PMCA2 protein. The cytoarchitecture of retinas from control and dfw2J mice was indistinguishable at the light microscope level. Suction electrode recordings revealed no difference in the sensitivity or amplitude of outer segment light responses of control and dfw2J rods. However, rod-mediated ERG b-wave responses in dfw2J mice were ∼45% smaller and significantly slower than those of control mice. Furthermore, recordings from individual rod bipolar cells showed that the sensitivity of transmission at the rod output synapse was reduced by ∼50%. No changes in the amplitude or timing of cone-mediated ERG responses were observed. These results suggest that PMCA2-mediated Ca2+ extrusion modulates the amplitude and timing of the high-sensitivity rod pathway to a much greater extent than that of the cone pathway.

C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor

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. DOI: http://dx.doi.org/10.7554/eLife.05981.001

Arrestin Competition Influences the Kinetics and Variability of the Single-Photon Responses of Mammalian Rod Photoreceptors

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.

Paucibacterial microbiome and resident DNA virome of the healthy conjunctiva

Purpose To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques. Methods Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses. Results Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65% of all conjunctiva samples tested. Conclusions Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.)

Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections

Detection of intraocular infections relies heavily on molecular diagnostics. A fundamental challenge is that only 100e300 ml of intraocular fluid can be safely obtained at any given time for diagnostic testing. The most widely available molecular diagnostic panel for infections in ophthalmology includes 4 separate pathogendirected polymerase chain reactions (PCRs): cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxoplasma gondii. Not surprisingly, more than 50% of all presumed intraocular infections fail to have a pathogen identified. Metagenomic deep sequencing has the potential to improve diagnostic yield as it is unbiased and hypothesis-free; it can theoretically detect all pathogens in a clinical sample. Previously, we demonstrated that unbiased RNA sequencing (RNA-seq) of intraocular fluid detects fungi, parasites, DNA and RNA viruses in uveitis patients. One obvious drawback regarding RNA-seq is that optimal RNA sequencing requires proper specimen handling, including either flash-freezing or immediate placement of the specimen on dry ice. Although commercial room-temperature RNA-preservatives may address this issue, practicing ophthalmologists may find these collection techniques impractical in an outpatient setting. For pathogens with DNA genomes, metagenomic DNA sequencing (DNA-seq) can circumvent this challenge, as DNA is more tolerant of ambient temperature. This study compares the performance of DNA-seq with conventional pathogen-directed PCRs to diagnose intraocular infections. De-identified archived vitreous samples received by the Proctor Foundation for pathogen-directed PCR testing from 2010e2015 were included. Samples were previously boiled and stored at 80 C. A total of 31 pathogen-positive reference samples and 36 pathogen-negative reference samples by Proctor PCRs were randomized and subjected to DNA-seq. The sensitivity and specificity of the Proctor’s pathogen-directed PCRs range from 85%e 100% and 98%e100%, respectively. The DNA-seq workflow included the following steps: 50 ml of each vitreous sample was used to isolate DNA using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) per the manufacturer’s recommendations, 5 ml of the extracted DNA were used to prepare libraries using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and amplified with 12 PCR cycles, and library size and concentration were determined as described. Samples were then sequenced to an average depth of 15 10 reads/sample on an Illumina HiSeq 4000 instrument using 125 nucleotide (nt) paired-end sequencing. Sequencing data were analyzed using a rapid computational pipeline developed in-house to classify sequencing reads and identify potential pathogens against the entire National Center for Biotechnology Information (NCBI) nt reference database. Any infectious agent that had 2 nonoverlapping reads to the reference pathogen genome was considered positive if it met both of the following criteria: (1) It is a pathogen known to be associated with infectious uveitis, and (2) reads from this pathogen were not present in the no-template (water only) control on the same run and library preparation. Discrepant samples were evaluated at the Clinical Laboratory Improvement Amendments (CLIA)-certified Stanford Clinical Microbiology and Clinical Virology laboratories, except for human T-cell leukemia virus type 1 (HTLV-1), which was confirmed at the University of California San Francisco using primers targeting the tax gene and subsequent amplicon sequencing (Elim Biopharmaceuticals, Inc, Hayward, CA). Laboratory personnel involved in sample preparation, analysis, and confirmatory testing were masked to the reference PCR results until all analyses were completed. Of the 31 positive-reference samples tested, 27 samples were identified correctly with DNA-seq (Fig 1A, Table S1, available at www.aaojournal.org). Three samples positive for T. gondii and 1 sample positive for VZV by directed-PCR were not detected by DNA-seq. The positivity of these 4 samples was confirmed by directed real-time PCRs. The cycle threshold for VZVwas 28.2, while the cycle thresholds for the 3 T. gondii samples ranged from 27e36.5. The positive agreement between DNA-seq and directed-PCR was 87%. Thirty-six archived vitreous samples that tested negative by all Proctor pathogen-directed PCRs (CMV, VZV, HSV, and T. gondii) were randomly selected for DNA-seq. Twenty eight of 36 samples yielded no additional pathogen (Fig 1B, Table S1, available at www.aaojournal.org), while 8 samples (22%) resulted in 6 additional pathogens either not detected or not tested with pathogen-directed PCRs. Those organisms included CMV, human herpesvirus 6 (HHV-6), HSV-2, HTLV-1, Klebsiella pneumoniae, and Candida dubliniensis. All of these organisms are known to be associated with infectious uveitis (Fig 1B). The results for HHV-6, HTLV-1, Klebsiella pneumoniae, and Candida dubliniensis were confirmed. Two samples tested positive for HSV-2 and CMV by DNA-seq were not confirmed by directed-PCR. Here, it was unclear if DNA-seq can achieve higher sensitivity for CMV and HSV-2 compared with PCR, or whether these were false-positive results by DNA-seq, as these samples yielded only 2 reads on DNA-seq. It should be noted that DNA-seq detected 100% of all samples that tested positive for CMV and HSV by PCR. An advantage of metagenomic deep sequencing is the ability to apply sequence information to infer the phenotypic behavior of the identified pathogen. We compared samples in which CMV sequences were adequately recovered for the UL54 and UL97 genes, coding for the DNA polymerase and phosphotransferase respectively, and compared with a CMV antiviral drug resistance database developed at Stanford University. Of the 7 samples analyzed, 3 had mutations in UL97 (phosphotransferase) that confer ganciclovir and valganciclovir resistance. Two samples were found to have C592G mutations, and 1 sample had both C592G and L595S mutations (Table S2, available at www.aaojournal.org). In summary, we showed that metagenomic DNA sequencing was highly concordant with pathogen-directed PCRs, despite nonideal sample handling conditions (boiled, long-term frozen). The unbiased nature of metagenomic DNA sequencing allowed an expanded scope of pathogen detection, including bacteria, fungal

Gut Microbial Diversity in Antibiotic-Naive Children After Systemic Antibiotic Exposure: A Randomized Controlled Trial

Background Antibiotic exposure can alter the gut microbiome. We evaluate the effects of azithromycin on the gut microbiome diversity of children from an antibiotic-naive community in Niger. Methods A population-based sample of 80 children aged 1-60 months in the Dosso region of Niger was randomized to receive a single dose of either oral azithromycin or placebo. Fecal samples were collected immediately before treatment and 5 days after treatment for 16S rRNA gene sequencing. The prespecified outcome was α-diversity (inverse Simpsons α-diversity index), with secondary outcomes of β and γ Simpsons and Shannons diversities. Results At 5 days after treatment, 40 children aged 1-60 months were analyzed in the azithromycin-treated group and 40 children in the placebo-treated group. Diversity of the gut microbiome was significantly lower in the treated group (inverse Simpsons α-diversity, 5.03; 95% confidence interval [CI], 4.08-6.14) than in the placebo group (6.91; 95% CI, 5.82-8.21; P = .03). Similarly, the Shannons α-diversity was lower in the treated group (10.60; 95% CI, 8.82-12.36) than the placebo group (15.42; 95% CI, 13.24-17.80; P = .004). Simpsons community-level (γ) diversity decreased with azithromycin exposure from 17.72 (95% CI, 13.80-20.21) to 10.10 (95% CI, 7.80-11.40; P = .00008), although β-diversity was not significantly reduced (2.56, 95% CI, 1.88-3.12; to 2.01, 95% CI, 1.46-2.51; P = .26). Conclusions Oral administration of azithromycin definitively decreases the diversity of the gut microbiome of children in an antibiotic-naive community. Clinical Trials Registration NCT02048007.

Retinal neovascularization and endogenous fungal endophthalmitis in intravenous drug users.

Intravenous heroin abuse in persons aged 12 or older has nearly doubled between the years of 2002 and 2012.1 Intravenous drug users (IVDU) are a high-risk group for developing endogenous fungal endophthalmitis (EFE), with Candida albicans being the most commonly identified causative organism.2 EFE is a potentially blinding disease in which the causative organism reaches the eye through hematogenous dissemination. The frequency of chorioretinitis and endophthalmitis in patients with documented fungemia ranges from 2–26% and 0–6%, respectively.3 Factors contributing to poor visual outcomes include macular involvement, subretinal and choroidal neovascularization, retinal detachment, and insufficient or delayed treatment.4,5 However, to our knowledge, this is the first case series characterizing retinal neovascularization as a potentially blinding complication of EFE in IVDU.

Metagenomic deep sequencing of aqueous fluid detects intraocular lymphomas

Introduction Currently, the detection of pathogens or mutations associated with intraocular lymphomas heavily relies on prespecified, directed PCRs. With metagenomic deep sequencing (MDS), an unbiased high-throughput sequencing approach, all pathogens as well as all mutations present in the host’s genome can be detected in the same small amount of ocular fluid. Methods In this cross-sectional case series, aqueous fluid samples from two patients were submitted to MDS to identify pathogens as well as common and rare cancer mutations. Results MDS of aqueous fluid from the first patient with vitreal lymphoma revealed the presence of both Epstein-Barr virus (HHV-4/EBV) and human herpes virus 8 (HHV-8) RNA. Aqueous fluid from the second patient with intraocular B-cell lymphoma demonstrated a less common mutation in the MYD88 gene associated with B-cell lymphoma. Conclusion MDS detects pathogens that, in some instances, may drive the development of intraocular lymphomas. Moreover, MDS is able to identify both common and rare mutations associated with lymphomas.