Violet-light suppression of thermogenesis by opsin 5 hypothalamic neurons.

The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.

Endophthalmitis Rate in Immediately Sequential versus Delayed Sequential Bilateral Cataract Surgery within the Intelligent Research in Sight (IRIS®) Registry Data.

PURPOSE: To compare the rate of postoperative endophthalmitis after immediately sequential bilateral cataract surgery (ISBCS) versus delayed sequential bilateral cataract surgery (DSBCS) using the American Academy of Ophthalmology Intelligent Research in Sight (IRIS®) Registry database. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients in the IRIS Registry who underwent cataract surgery from 2013 through 2018. METHODS: Patients who underwent cataract surgery were divided into 2 groups: (1) ISBCS and (2) DSBCS (second-eye surgery ≥1 day after the first-eye surgery) or unilateral surgery. Postoperative endophthalmitis was defined as endophthalmitis occurring within 4 weeks of surgery by International Classification of Diseases (ICD) code and ICD code with additional clinical criteria. MAIN OUTCOME MEASURES: Rate of postoperative endophthalmitis. RESULTS: Of 5 573 639 IRIS Registry patients who underwent cataract extraction, 165 609 underwent ISBCS, and 5 408 030 underwent DSBCS or unilateral surgery (3 695 440 DSBCS, 1 712 590 unilateral surgery only). A total of 3102 participants (0.056%) met study criteria of postoperative endophthalmitis with supporting clinical findings. The rates of endophthalmitis in either surgery eye between the 2 surgery groups were similar (0.059% in the ISBCS group vs. 0.056% in the DSBCS or unilateral group; P = 0.53). Although the incidence of endophthalmitis was slightly higher in the ISBCS group compared with the DSBCS or unilateral group, the odds ratio did not reach statistical significance (1.08; 95% confidence interval, 0.87-1.31; P = 0.47) after adjusting for age, sex, race, insurance status, and comorbid eye disease. Seven cases of bilateral endophthalmitis with supporting clinical data in the DSBCS group and no cases in the ISBCS group were identified. CONCLUSIONS: Risk of postoperative endophthalmitis was not statistically significantly different between patients who underwent ISBCS and DSBCS or unilateral cataract surgery.

Systemic prime exacerbates the ocular immune response to heat-killed Mycobacterium tuberculosis.

Post-infectious uveitis describes the condition of chronic immune mediated ocular inflammation associated with pathogens such as Mycobacterium tuberculosis (Mtb). Mtb associated post-infectious uveitis can be modeled in mice by intravitreal injection of heat-killed Mtb (HKMtb). To better understand how prior systemic exposure to the pathogen alters the local immune response to Mtb, we used flow cytometry and multiplex ELISAs to compare ocular responses to intravitreal HKMtb in the presence or absence of a systemic "prime" of HKMtb. Priming resulted in exacerbation of local inflammation with significantly increased clinical and histologic inflammation scores and increased vitreous cytokines concentrations one day after intravitreal injection of HKMtb. Seven days after injection, uveitis in unprimed animals had largely resolved. In contrast in primed animals, clinical signs of chronic inflammation were associated with a significant increase in the number of ocular T cells, NK cells, and Ly6Chi macrophages and increasing vitreous concentrations of IL-17, VEGF, MIG(CXCL9), IP-10(CXCL10), IL-12p40 and MIP-1α(CCL3). In mice lacking mature T and B cells (RAG2 deficient), the impact of priming on the ocular immune response was ameliorated with significantly lower vitreous cytokine concentrations and spontaneous resolution of uveitis. Altogether these results suggest that the ocular response to Mtb is exacerbated by prior systemic Mtb infection and chronic post-infectious uveitis is mediated by local production of cytokines and chemokines that amplify Th17 and Th1 responses. This mouse model of chronic Mtb associated uveitis will help elucidate mechanisms of disease in patients with post-infectious uveitis.

RESPONSE OF INFLAMMATORY CYSTOID MACULAR EDEMA TO TREATMENT USING ORAL ACETAZOLAMIDE.

PURPOSE: To determine the treatment effect of oral acetazolamide on refractory inflammatory macular edema. METHODS: A retrospective review of identified patients with uveitic or pseudophakic macular edema treated using acetazolamide between 2007 and 2014. Visual acuity and central macular subfield thickness was determined at baseline and at first follow-up. Baseline optical coherence tomography features were analyzed as predictors of acetazolamide response. RESULTS: Sixteen patients (19 eyes) of 61 screened met all criteria. Mean age was 57.9 years (19.7-81.1). The most common diagnosis was idiopathic uveitis (n = 6, 31.6%). Mean uveitis duration was 4.4 years (0.2-27.5). Average central macular subfield thickness decreased significantly (from 471.8 ± 110.6 µm to 358.3 ± 50.4 µm) (P < 0.0001). Average visual acuity (logarithm of the minimum angle of resolution) improved significantly from 20/54 (0.43 ± 0.25) to 20/37 (0.27 ± 0.16) (P = 0.003). Pretreatment optical coherence tomographies demonstrated intraretinal fluid (n = 19, 100%), subretinal fluid (n = 8, 42.1%), epiretinal membrane (n = 13, 68.3%), and vitreomacular traction (n = 1, 5.2%). No optical coherence tomography characteristic was predictive of a response to therapy. CONCLUSION: There is a significant benefit to vision and central macular subfield thickness after acetazolamide treatment in patients with inflammatory macular edema. In patients with refractory inflammatory macular edema, treatment using acetazolamide can provide anatomical and visual benefit without corticosteroid-related adverse effects.

Determinants of Outcomes of Adenoviral Keratoconjunctivitis.

PURPOSE: To determine host and pathogen factors predictive of outcomes in a large clinical cohort with keratoconjunctivitis. DESIGN: Retrospective analyses of the clinical and molecular data from a randomized, controlled, masked trial for auricloscene for keratoconjunctivitis (NVC-422 phase IIB, NovaBay; clinicaltrials.gov identifier, NCT01877694). PARTICIPANTS: Five hundred participants from United States, India, Brazil, and Sri Lanka with clinical diagnosis of keratoconjunctivitis and positive rapid test results for adenovirus. METHODS: Clinical signs and symptoms and bilateral conjunctival swabs were obtained on days 1, 3, 6, 11, and 18. Polymerase chain reaction (PCR) analysis was performed to detect and quantify adenovirus in all samples. Regression models were used to evaluate the association of various variables with keratoconjunctivitis outcomes. Time to resolution of each symptom or sign was assessed by adenoviral species with Cox regression. MAIN OUTCOME MEASURES: The difference in composite scores of clinical signs between days 1 and 18, mean visual acuity change between days 1 and 18, and time to resolution of each symptom or sign. RESULTS: Of 500 participants, 390 (78%) showed evidence of adenovirus by PCR. Among adenovirus-positive participants, adenovirus D species was most common (63% of total cases), but a total of 4 species and 21 different types of adenovirus were detected. Adenovirus D was associated with more severe signs and symptoms, a higher rate of subepithelial infiltrate development, and a slower decline in viral load compared with all other adenovirus species. The clinical courses of all patients with non-adenovirus D species infection and adenovirus-negative keratoconjunctivitis were similar. Mean change in visual acuity between days 1 and 18 was a gain of 1.9 letters; worse visual outcome was associated with older age. CONCLUSIONS: A substantial proportion of keratoconjunctivitis is not associated with a detectable adenovirus. The clinical course of those with adenovirus D keratoconjunctivitis is significantly more severe than those with non-adenovirus D species infections or adenovirus-negative keratoconjunctivitis; high viral load at presentation and non-United States origin of participants is associated with poorer clinical outcome.

Neuropsin (OPN5)-mediated photoentrainment of local circadian oscillators in mammalian retina and cornea.

The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength-sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.

Local photic entrainment of the retinal circadian oscillator in the absence of rods, cones, and melanopsin.

Synchronization of the mammalian master circadian pacemaker to the daily light/dark cycle is mediated exclusively through retinal photoreceptors. The mammalian retina itself is also a self-sustained circadian oscillator. Here we report that the retinal molecular circadian clock can be entrained by lighting cycles in vitro, but that rods, cones, and melanopsin (Opn4) are not required for this entrainment. In vivo, retinas of Opn4(-/-);rd1/rd1 mice synchronize to light/dark cycles regardless of the phase of the master circadian pacemakers of the suprachiasmatic nuclei or the behavior of the animal. These data demonstrate that the retina uses a separate mechanism for local entrainment of its circadian clock than for entrainment of organism-level rhythmicity.

Scalable metagenomics alignment research tool (SMART): a scalable, rapid, and complete search heuristic for the classification of metagenomic sequences from complex sequence populations.

BACKGROUND: Next generation sequencing technology has enabled characterization of metagenomics through massively parallel genomic DNA sequencing. The complexity and diversity of environmental samples such as the human gut microflora, combined with the sustained exponential growth in sequencing capacity, has led to the challenge of identifying microbial organisms by DNA sequence. We sought to validate a Scalable Metagenomics Alignment Research Tool (SMART), a novel searching heuristic for shotgun metagenomics sequencing results. RESULTS: After retrieving all genomic DNA sequences from the NCBI GenBank, over 1 × 10(11) base pairs of 3.3 × 10(6) sequences from 9.25 × 10(5) species were indexed using 4 base pair hashtable shards. A MapReduce searching strategy was used to distribute the search workload in a computing cluster environment. In addition, a one base pair permutation algorithm was used to account for single nucleotide polymorphisms and sequencing errors. Simulated datasets used to evaluate Kraken, a similar metagenomics classification tool, were used to measure and compare precision and accuracy. Finally using a same set of training sequences we compared Kraken, CLARK, and SMART within the same computing environment. Utilizing 12 computational nodes, we completed the classification of all datasets in under 10 min each using exact matching with an average throughput of over 1.95 × 10(6) reads classified per minute. With permutation matching, we achieved sensitivity greater than 83 % and precision greater than 94 % with simulated datasets at the species classification level. We demonstrated the application of this technique applied to conjunctival and gut microbiome metagenomics sequencing results. In our head to head comparison, SMART and CLARK had similar accuracy gains over Kraken at the species classification level, but SMART required approximately half the amount of RAM of CLARK. CONCLUSIONS: SMART is the first scalable, efficient, and rapid metagenomics classification algorithm capable of matching against all the species and sequences present in the NCBI GenBank and allows for a single step classification of microorganisms as well as large plant, mammalian, or invertebrate genomes from which the metagenomic sample may have been derived.

Evaluating Access to Eye Care in the Contiguous United States by Calculated Driving Time in the United States Medicare Population.

PURPOSE: To quantify the proximity to eye care in the contiguous United States by calculating driving routes and driving time using a census-based approach. DESIGN: Cross-sectional study based on United States (US) census data, Medicare payment data, and OpenStreetMap. PARTICIPANTS: 2010 US census survey respondents older than 65 years. METHODS: For each state in the United States, the addresses of all practicing ophthalmologists and optometrists were obtained from the 2012 Medicare Provider Utilization and Payment Data from the Centers for Medicare and Medicaid Services (CMS). The US census data from 2010 then were used to calculate the geolocation of the US population at the block group level and the number of people older than 65 years in each location. Geometries and driving speed limits of every road, street, and highway in the United States from the OpenStreetMap project were used to calculate the exact driving distance and driving time to the nearest eye care provider. MAIN OUTCOME MEASURES: Driving time and driving distance to the nearest optometrist and ophthalmologist per state. RESULTS: Driving times for 3.79×107 persons were calculated using a total of 3.88×107 available roads for the 25 508 optometrists and 17 071 ophthalmologists registered with the CMS. Nationally, the median driving times to the nearest optometrist and ophthalmologist were 2.91 and 4.52 minutes, respectively. Ninety percent of the population lives within a 13.66- and 25.21-minute drive, respectively, to the nearest optometrist and ophthalmologist. CONCLUSIONS: While there are regional variations, overall more than 90% of the US Medicare beneficiary population lives within a 30-minute drive of an ophthalmologist and within 15 minutes of an optometrist.

Ramshackle (Brwd3) promotes light-induced ubiquitylation of Drosophila Cryptochrome by DDB1-CUL4-ROC1 E3 ligase complex.

Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. It resets the circadian clock by promoting light-induced degradation of the clock proteins Timeless and Period, as well as its own proteolysis. The E3 ligases that ubiquitylate Timeless and Period before degradation are known and it is known that Drosophila (d) CRY is degraded by the ubiquitin-proteasome system as well. To identify the E3 ligase for dCRY we screened candidates in S2 cells by RNAi. Knockdown of each of the 25 putative F-box proteins identified by bioinformatics did not attenuate the light-induced degradation of dCRY. However, knockdown of a WD40 protein, Bromodomain and WD repeat domain containing 3 (Brwd3) (CG31132/Ramshackle) caused strong attenuation of dCRY degradation following light exposure. We found that BRWD3 functions as a Damage-specific DNA binding protein 1 (DDB1)- and CULLIN (CUL)4-associated factor in a Cullin4-RING Finger E3 Ligase (CRL4) that mediates light-dependent binding of dCRY to CUL4-ROC1-DDB1-BRWD3, inducing ubiquitylation of dCRY and its light-induced degradation. Thus, this study identifies a light-activated E3 ligase complex essential for light-mediated CRY degradation in Drosophila cells.

Identification of torque teno virus in culture-negative endophthalmitis by representational deep DNA sequencing.

PURPOSE: To test the hypothesis that uncultured organisms may be present in cases of culture-negative endophthalmitis by use of deep DNA sequencing of vitreous biopsies. DESIGN: Single-center, consecutive, prospective, observational study. PARTICIPANTS: Aqueous or vitreous biopsies from 21 consecutive patients presenting with presumed infectious endophthalmitis and 7 vitreous samples from patients undergoing surgery for noninfectious retinal disorders. METHODS: Traditional bacterial and fungal culture, 16S quantitative polymerase chain reaction (qPCR), and a representational deep-sequencing method (biome representational in silico karyotyping [BRiSK]) were applied in parallel to samples to identify DNA sequences corresponding to potential pathogens. MAIN OUTCOME MEASURES: Presence of potential pathogen DNA in ocular samples. RESULTS: Zero of 7 control eyes undergoing routine vitreous surgery yielded positive results for bacteria or virus by culture or 16S polymerase chain reaction (PCR). A total of 14 of the 21 samples (66.7%) from eyes harboring suspected infectious endophthalmitis were culture-positive, the most common being Staphylococcal and Streptococcal species. There was good agreement among culture, 16S bacterial PCR, and BRiSK methodologies for culture-positive cases (Fleiss' kappa of 0.621). 16S PCR did not yield a recognizable pathogen sequence in any culture-negative sample, whereas BRiSK suggested the presence of Streptococcus in 1 culture-negative sample. With the use of BRiSK, 57.1% of culture-positive and 100% of culture-negative samples demonstrated the presence of torque teno virus (TTV) sequences, compared with none in the controls (P=0.0005, Fisher exact test). The presence of TTV viral DNA was confirmed in 7 cases by qPCR. No other known viruses or potential pathogens were identified in these samples. CONCLUSIONS: Culture, 16S qPCR, and BRiSK provide complementary information in presumed infectious endophthalmitis. The majority of culture-negative endophthalmitis samples did not contain significant levels of bacterial DNA. "Culture negativity" does not seem to be due to failure of growth of fastidious bacteria. The small DNA virus TTV was unexpectedly found in all culture-negative samples and some culture-positive samples. This study cannot distinguish whether TTV is a direct intraocular pathogen, an adjuvant for inflammation, a general marker of inflammation, or a commensal virus but provides a testable hypothesis for a pathogenic mechanism in culture-negative endophthalmitis.

Emerging techniques for pathogen discovery in endophthalmitis.

PURPOSE OF REVIEW: Despite the inability to detect certain organisms and relatively low yield, microbial culture is the current gold standard for the diagnosis of most intraocular infections. Research on alternative molecular diagnostic methods has produced an array of strategies that augment and improve pathogen detection. This review summarizes the most recent literature on this topic. RECENT FINDINGS: The yield of traditional microbial culture has not improved since the Endophthalmitis Vitrectomy Study results were published 20 years ago. Advances in PCR methods have enabled quantification of pathogen load and screening for multiple organisms at once. More recently, deep sequencing techniques allow highly sensitive detection of any DNA-based life form in a specimen. This offers the promise of not only improved detection of traditional organisms but can also identify organisms not previously associated with endophthalmitis. SUMMARY: Molecular diagnostic methods enhance the results of microbial culture and may become the new standard in the diagnosis of intraocular infections.

Biome representational in silico karyotyping.

Metagenomic characterization of complex biomes remains challenging. Here we describe a modification of digital karyotyping-biome representational in silico karyotyping (BRISK)-as a general technique for analyzing a defined representation of all DNA present in a sample. BRISK utilizes a Type IIB DNA restriction enzyme to create a defined representation of 27-mer DNAs in a sample. Massively parallel sequencing of this representation allows for construction of high-resolution karyotypes and identification of multiple species within a biome. Application to normal human tissue demonstrated linear recovery of tags by chromosome. We apply this technique to the biome of the oral mucosa and find that greater than 25% of recovered DNA is nonhuman. DNA from 41 microbial species could be identified from oral mucosa of two subjects. Of recovered nonhuman sequences, fewer than 30% are currently annotated. We characterized seven prevalent unknown sequences by chromosome walking and find these represent novel microbial sequences including two likely derived from novel phage genomes. Application of BRISK to archival tissue from a nasopharyngeal carcinoma resulted in identification of Epstein-Barr virus infection. These results suggest that BRISK is a powerful technique for the analysis of complex microbiomes and potentially for pathogen discovery.