Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.

Large-scale deep tissue voltage imaging with targeted illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by the tremendous advances in genetically encoded voltage indicators (GEVIs). However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines signal-to-noise ratio (SNR) and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating, while also maximizing signal detection efficiency. The resulting benefits in SNR and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different GEVI classes.

Patient factors associated with appointment non-attendance at an ophthalmology department in Aotearoa New Zealand.

AIM: Appointment non-attendance is a problem for medical outpatient clinics, which can result in interruption of continuity of care and poor health outcomes for patients. Furthermore, non-attendance creates a significant economic burden to the health sector. This study aimed to identify factors that are associated with appointment non-attendance in a large public ophthalmology clinic in Aotearoa New Zealand. METHODS: This study was a retrospective analysis of clinic non-attendance within Auckland District Health Board's (DHB) Ophthalmology Department between 1 January 2018 to 31 December 2019. Demographic data collected included: age, gender and ethnicity. Deprivation Index was calculated. Appointments were classified as new patients and follow-ups, and acute or routine. Categorical and continuous variables were analysed using logistic regression to assess likelihood of non-attendance. The research team's expertise and capacity align with the CONSIDER statement guidelines for Indigenous health and research. RESULTS: In total, 52,512 patients were scheduled to attend 227,028 outpatient visits, of which 20,580 visits (9.1%) were not attended. Median age of patients who received one or more scheduled appointments were 66.1 years (interquartile range [IQR] 46.9-77.9). Fifty-one point seven percent of patients were female. Ethnicity comprised 55.0% European, 7.9% Maori, 13.5% Pacific peoples, 20.6% Asian and 3.1% Other. Multivariate logistic regression analysis for all appointments showed that males (odds ratio [OR] 1.15 p<0.001), younger patients (OR 0.99 p<0.001), Maori (OR 2.69 p<0.001), Pacific peoples (OR 2.82 p<0.001), higher deprivation status (OR 1.06 p<0.001), new patient appointments (OR 1.61 p<0.001) and patients referred to acute clinics (OR 1.22 p<0.001) were more likely to not attend appointments. CONCLUSIONS: Maori and Pacific peoples disproportionately experience higher rates of appointment non-attendance. Further investigation of access barriers will enable Aotearoa New Zealand health strategy planning to develop targeted interventions addressing unmet patient needs of at-risk groups.

Rhegmatogenous retinal detachment presentation and surgery in uveitic eyes.

BACKGROUND/AIMS: To explore the occurrence, uveitis activity, features, rate of proliferative vitreoretinopathy (PVR) and outcomes following rhegmatogenous retinal detachment (RRD) in a large tertiary referral uveitis service. METHODS: Retrospective analysis of subjects attending between 2008 and 2019. Multivariate analysis of risk factors for RRD was calculated. Nelson-Aalen plots were used to demonstrate cumulative risk of RRD. Outcomes of RRD surgery and prognostic indicators were analysed. RESULTS: Two thousand four hundred and forty-seven (2447) subjects (3516 eyes) with uveitis included. The mean follow-up was 5.7 years (19 767 eye-years); 56 eyes developed a RRD (1.6%). Thirty-two eyes had surgery in our unit. Risk factors for RRD were posterior uveitis or panuveitis (HR 3.386, p<0.001), male gender (HR 2.045, p=0.029) and infectious aetiology (HR 1.942, p=0.044). PVR was present in six (18.8%) eyes at presentation, and a further four (12.5%) developed it after the primary surgery. Final follow-up data showed 16 (50%) moderate or severe visual loss, although 29 (90.6%) had anatomical reattachment without oil in situ. CONCLUSIONS: There is a high rate of RRD in uveitis eyes. This is accompanied by high rates of PVR and redetachment. Anatomical success was high, but visual outcomes remain unpredictable.

Iris melanocytic tumours in New Zealand/Aotearoa: presentation, management and outcome in a high UV exposure environment.

BACKGROUND/OBJECTIVES: Iris melanoma, a rare intraocular malignancy, represents the smallest subgroup of uveal melanoma. This first, comprehensive study of iris melanocytic lesions in the high ultraviolet environment in New Zealand/ Aotearoa (NZ) examines diagnosis, management and outcomes. SUBJECTS/METHODS: Retrospective study of iris melanocytic tumours referred to tertiary referral centres in Auckland, NZ, over 20 years (1999-2018). Data analysed include demographics, tumour characteristics, histology, genetic analyses, treatment modalities, recurrence, metastasis, 5-year and overall survival. RESULTS: Cohort (N = 51) was predominantly NZ European (98.0%) with no indigenous Maori, or Pasifika. Median age at presentation was 58 years. Tumours involved a median of two clock hours of iris. The posterior tumour margin extended to the anterior chamber angle in 22 patients (45.8%). Management included initial observation 54.9%, iridectomy/excision biopsy 29.4%, irido-cyclectomy 7.8%, plaque radiotherapy 7.8%, proton beam radiotherapy 7.8%, and ultimately enucleation 17.6%. Histology was performed in 19 cases (37%) with 16 confirmed melanomas (84%). Mean follow-up 4.2 years with median visual acuity of 6/7.5 two years post intervention. Melanoma-related metastasis and mortality occurred in two cases with five-year melanoma-related mortality of 2.0%. CONCLUSION: In a climate with high ultraviolet exposure iris melanocytic tumours occurred almost exclusively in NZ Europeans, however, the majority of cases were category T1, possibly reflecting early diagnosis in the NZ health system. Nonetheless, >50% underwent surgery or radiotherapy, often utilising more than one modality. A high index of suspicion and early referral of iris melanocytic lesions should be considered in regions with high UV exposure.

Comparative Outcomes of Primary Vitrectomy Versus Tap and Inject for Endophthalmitis Following Phacoemulsification Cataract Surgery.

PURPOSE: To compare functional and anatomical outcomes, rates of culture positivity and number of procedures in eyes with endophthalmitis following phacoemulsification surgery, treated with either primary vitrectomy and intravitreal antibiotics or vitreous tap and antibiotic injection (T&I). METHODS: Patients developing endophthalmitis after phacoemulsification surgery between 2007 and 2016 were identified, and outcomes were compared between the two treatment groups. RESULTS: 19 patients underwent a primary vitrectomy and 22 underwent a T&I. There was a significant improvement in visual acuity after T&I (p=.003) and primary vitrectomy (p=.00005). The median improvement in visual acuity was significantly greater for the primary vitrectomy group than the T&I group (p=.024). 64% of eyes were culture positive with the initial T&I, and 63% with primary vitrectomy (p=1.00). Two eyes initially culture negative with a T&I, and three eyes that were culture positive with a T&I were subsequently culture positive with a vitrectomy 24-72 hours later. 68% of patients who underwent a T&I required an additional procedure, compared to 26% of the vitrectomy group (p=.01). The T&I group underwent a mean of 2.3 procedures each, and the primary vitrectomy group underwent 1.5 (p=.03). CONCLUSIONS: Eyes with endophthalmitis treated with a primary vitrectomy demonstrated greater visual improvement and needed fewer procedures than those initially treated with a T&I. Viable bacteria were only seen in subsequent procedures in the T&I group, indicating that primary vitrectomy was superior at sterilizing the eye.

Enhancing Maori and Pasifika graduate interest in ophthalmology surgical training in New Zealand/Aotearoa: Barriers and opportunities.

IMPORTANCE: Improving the representation of indigenous ophthalmologists in New Zealand. BACKGROUND: Maori, indigenous to New Zealand/Aotearoa and Pacific Peoples indigenous to Pacific Island Nations living in New Zealand, experience poorer health outcomes across several ophthalmic conditions. The Royal Australian and New Zealand College of Ophthalmologists have identified indigenous workforce development as a priority. DESIGN: Mixed-methods study, utilizing retrospective analysis of Medical Schools Outcomes Database and Longitudinal Tracking Project responses, and prospective interviews with Maori/Pasifika medical graduates. PARTICIPANTS: This study involved 64 medical graduates from the University of Auckland (UoA) and the University of Otago, and six Maori/Pasifika medical postgraduates in New Zealand. METHODS: Retrospective analysis of medical graduate responses who ranked ophthalmology among their top-three preferred specialties in the Medical Schools Outcomes Database and Longitudinal Tracking Project. Prospective semi-structured interviews with Maori/Pasifika medical postgraduates. MAIN OUTCOME MEASURES: Specialty training influencing factors and prevocational ophthalmology experience. RESULTS: A total of 64 (6.7%) out of 954 medical graduates from the UoA and University of Otago ranked ophthalmology among their top-three preferred training specialties (2012-2017). Of the 64 graduates, six (9.3%) identified as Maori/Pasifika. No significant difference in influencing factors between Maori/Pasifika and non-Maori/Pasifika students was identified. Both groups ranked intellectual content, procedural skills, specialty exposure and mentorship as highly influential. Qualitative interviews with Maori/Pasifika graduates highlighted positive experiences in ophthalmology but limited exposure overall. Negative anecdotes and unclear training pathways discouraged Maori/Pasifika interest in Ophthalmology training. CONCLUSIONS AND RELEVANCE: Maori/Pasifika graduate interest in ophthalmology training was relatively low. Valuable insights include enhancing specialty exposure, mentor development, promoting Maori/Pasifika connections and clarifying training pathways for future graduates.

CUTICULAR DRUSEN IN AN INDIGENOUS AUSTRALIAN.

BACKGROUND/PURPOSE: To report a case of cuticular drusen in an indigenous Australian. METHODS: A 37-year-old indigenous (aboriginal) Australian woman from a remote Western Australian town presented with a 2-month history of vision loss. Clinical history, examination, and multimodal retinal imaging data from spectral domain optical coherence tomography, fundus autofluorescence, fluorescein angiography, and indocyanine green angiography were analyzed. RESULTS: Multimodal imaging confirmed cuticular drusen complicated by a right choroidal neovascularization with pigment epithelial detachment and a left foveal vitelliform lesion. An unusual "negative-staining" pattern was noted on late phase indocyanine green angiography and this spared the regions affected by cuticular drusen. CONCLUSION: To the best of our knowledge, this is the first published report of cuticular drusen in an indigenous Australian. We hypothesize that this may be secondary to ancestral mixing of the gene pool. An unusual staining pattern witnessed on indocyanine green angiography suggests widespread disturbance of lipoprotein deposition in the Bruch membrane.

Vacuum Selection from Cosmology on Networks of String Geometries.

We introduce network science as a framework for studying the string landscape. Two large networks of string geometries are constructed, where nodes are extra-dimensional six-manifolds and edges represent topological transitions between them. We show that a standard bubble cosmology model on the networks has late-time behavior determined by the largest eigenvector of -(L+D), where L and D are the Laplacian and degree matrices of the networks, which provides a dynamical mechanism for vacuum selection in the string landscape.

The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo.

UNLABELLED: Pharmacologically-induced activation of replication competent proviruses from latency in the presence of antiretroviral treatment (ART) has been proposed as a step towards curing HIV-1 infection. However, until now, approaches to reverse HIV-1 latency in humans have yielded mixed results. Here, we report a proof-of-concept phase Ib/IIa trial where 6 aviremic HIV-1 infected adults received intravenous 5 mg/m2 romidepsin (Celgene) once weekly for 3 weeks while maintaining ART. Lymphocyte histone H3 acetylation, a cellular measure of the pharmacodynamic response to romidepsin, increased rapidly (maximum fold range: 3.7­7.7 relative to baseline) within the first hours following each romidepsin administration. Concurrently, HIV-1 transcription quantified as copies of cell-associated un-spliced HIV-1 RNA increased significantly from baseline during treatment (range of fold-increase: 2.4­5.0; p = 0.03). Plasma HIV-1 RNA increased from <20 copies/mL at baseline to readily quantifiable levels at multiple post-infusion time-points in 5 of 6 patients (range 46­103 copies/mL following the second infusion, p = 0.04). Importantly, romidepsin did not decrease the number of HIV-specific T cells or inhibit T cell cytokine production. Adverse events (all grade 1­2) were consistent with the known side effects of romidepsin. In conclusion, romidepsin safely induced HIV-1 transcription resulting in plasma HIV-1 RNA that was readily detected with standard commercial assays demonstrating that significant reversal of HIV-1 latency in vivo is possible without blunting T cell-mediated immune responses. These finding have major implications for future trials aiming to eradicate the HIV-1 reservoir. TRIAL REGISTRATION: clinicaltrials.gov NTC02092116.

Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

OBJECTIVES: To determine the contribution of peripheral blood mononuclear cells' (PBMCs) HIV DNA levels to HIV-associated dementia (HAD) and non-demented HIV-associated neurocognitive disorders (HAND) in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART). METHODS: Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL) were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points. RESULTS: At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03). Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05). Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC). Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04), and higher in those with no ART treatment during HIV infection 1st year (p = .03). Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03), and motor-coordination (p = .02-.12) to a lesser extent. CONCLUSIONS: PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

Early antiretroviral therapy with raltegravir generates sustained reductions in HIV reservoirs but not lower T-cell activation levels.

OBJECTIVE: The initiation of antiretroviral therapy (ART) during primary infection may offer clinical benefits for HIV-infected individuals by reducing HIV DNA reservoir size and chronic T-cell activation. Current evidence for the advantages of early ART, however, are mostly derived from cross-sectional studies, with the long-term benefits yet to be ascertained. DESIGN/METHODS: We conducted an open-label, nonrandomized study, monitoring for 3 years: plasma viral load (pVL), T-cell phenotypes, and peripheral CD4(+) T-cell associated total, integrated and 2-long terminal repeat HIV DNA species. The study included 16 treatment-naive individuals initiating ART with raltegravir and Truvada during either primary (PHI, n = 8) or chronic (CHI, n = 8) HIV infection. RESULTS: ART initiated during PHI compared with CHI generated significant reductions of peripheral CD4(+) T-cell HIV DNA reservoirs that were sustained for 3 years of therapy. Median log10 HIV DNA copies/10(6) CD4(+) T cells at the final visit: total; CHI = 3.23 > PHI = 2.72, P < 0.01; integrated; CHI = 2.64 > PHI = 1.77, P < 0.01. Similar trends were observed for pVL, however, did not reach significance: log10 HIV RNA copies/ml plasma at the final visit: CHI = 1.3 ≥ PHI = 0.39, P = 0.08. Both cohorts displayed similar and elevated levels of CD38/HLA-DR coexpression on CD4(+) and CD8(+) T cells relative to uninfected healthy controls. CONCLUSION: The reduction in HIV DNA reservoirs generated by the early initiation of ART was sustained for 3 years of therapy. Although the PHI cohort trended to lower levels of pVL, and pVL was associated with CD8(+) T-cell activation, no differences in T-cell activation were observed between the PHI and CHI groups.

Characterization of Barmah Forest virus pathogenesis in a mouse model.

Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in human BFV infection are very similar to those of RRV. Although RRV disease has been studied extensively, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis, and to identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Here, to the best of our knowledge, we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to that of RRV. In comparison with RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-α, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge, this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understand disease pathogenesis. With an increasing number of BFV cases occurring annually, a better understanding of the disease mechanisms is essential for future therapeutic development.

Water and organic synthesis: a focus on the in-water and on-water border. Reversal of the in-water Breslow hydrophobic enhancement of the normal endo-effect on crossing to on-water conditions for Huisgen cycloadditions with increasingly insoluble organic liquid and solid 2π-dipolarophiles.

Measurements of the endo/exo product ratios for Huisgen cycloadditions with a series of vinyl ketones, alkyl acrylates, and substituted styrenes as dipolarophiles with phthalazinium and pyridazinium dicyanomethanide 1,3-dipoles in acetonitrile and water show that as the reactions change from in-water (large hydrophobic enhancement of endo-products) to on-water, the hydrophobic enhancement of the endo-products is reduced and partially reversed (relative to acetonitrile). An expected increase of the endo-effect with increasing hydrophobic character of the dipolarophile is overcome by decreasing water solubility causing changeover to on-water conditions. On-water reactions do not show increased cycloaddition endo-effects (relative to organic solvents) as do in-water reactions.

The New Zealand National Eye Bank study: trends in the acquisition and storage of corneal tissue over the decade 2000 to 2009.

PURPOSE: To evaluate trends in the acquisition, storage, and utilization of donated corneal tissue in New Zealand, 2000 to 2009. METHODS: The New Zealand National Eye Bank records were analyzed for the decade January 2000 to December 2009. Variables analyzed included donor demographics (age, sex, and ethnicity), donor source, donor cause of death, death-to-preservation interval (DPI), corneal storage time, tissue contamination, endothelial assessment, cornea suitability for transplantation, and corneal tissue utilization. RESULTS: A total of 1268 eye donors were identified during the 10-year period. Overall, 36% (n = 457) were female and 64% male (n = 813). Median donor age was 67 years, and 23% of donors were younger than 50 years (range, 5-90 years). There was a decrease in donor age over the decade (P = 0.006). The median DPI was 18.5 hours. No relationship was identified between cornea suitability for transplantation and DPI (P = 0.28) or donor gender (P = 0.54). There was a low microbial contamination rate (1%). Human immunodeficiency virus, hepatitis B, or hepatitis C serology was positive in 48 donors (4%). Overall, 90% of corneas were suitable for transplantation with a high utilization rate (88%). A novel association was identified between male sex and lower corneal endothelial cell density (P = 0.03). CONCLUSIONS: This New Zealand National Eye Bank analysis identified trends in the acquisition, storage, and utilization of donated corneal tissue throughout New Zealand over the past decade and provides valuable additional information to the international eye bank data.

Trends in the distribution of donor corneal tissue and indications for corneal transplantation: the New Zealand National Eye Bank Study 2000-2009.

BACKGROUND: To investigate the indications for corneal transplantation and the distribution of donor corneal tissue in New Zealand. DESIGN: Analysis of the prospective database of the New Zealand National Eye Bank. PARTICIPANTS: A total of 2205 corneal transplants were assessed. METHODS: New Zealand National Eye Bank records were analysed for the decade 2000-2009. MAIN OUTCOME MEASURES: Variables analysed included donor corneal tissue distribution (including public and private sectors), indications for transplantation, donor corneal tissue recipient demographics (age and gender) and corneal transplantation type. RESULTS: An average of 220 corneal transplants were performed each year over the 10-year period (n=2205). The median recipient age was 45years (range 3 to 102years) and 54.0% of recipients were male. In total 71.8% of transplants were performed in the public health sector. Surgeons in the Auckland metropolitan area performed 47.2% of all corneal transplants. The most common indications for corneal transplantation were: keratoconus (41.1%), repeat transplant (17.0%), aphakic/pseudophakic bullous keratopathy (13.9%), corneal dystrophy (10.7%), keratitis (7.9%) and trauma (3.7%). Overall, penetrating keratoplasty accounted for 90.7% of all corneal transplants, however, during the latter half of the study there was a progressive shift in transplantation type, with deep anterior lamellar keratoplasty and Descemet's stripping endothelial keratoplasty combined accounting for 32.3% of all transplants in the final year of the study period. CONCLUSIONS: This New Zealand National Eye Bank study provides valuable data regarding the indications for corneal transplantation, transplant recipient demographics and changes in transplantation type in New Zealand over the past decade.

The influence of water on the rates of 1,3-dipolar cycloaddition reactions: trigger points for exponential rate increases in water-organic solvent mixtures. Water-super versus water-normal dipolarophiles.

The nature of the rate enhancements caused by gradually increasing the mole fraction of water in the solvent (from 0 to 1) for the cycloaddition reactions of pyridazinium-dicyanomethanide 1,3-dipole, 2, with the dipolarophiles ethyl vinyl ketone (a water-super dipolarophile) and methyl acrylate (a water-normal dipolarophile) in the organic solvents acetonitrile, acetone, methanol, ethanol, and tert-butyl alcohol at 37 degrees C are explored. In each case as the mole fraction of water surpassed ca. 0.9, exponential rate enhancements were triggered. When methanol replaced water, the rate enhancements were smaller, and no triggering effect was observed. The dramatic rate enhancement triggered for the water-super dipolarophile was significantly reduced as the temperature was raised in the range 29-64 degrees C. The results suggest that a dominant hydrogen-bonding effect operates in water-induced rate enhancements of 1,3-dipolar cycloaddition reactions with water-super dipolarophiles as well as the hydrophobic effect. The hydrogen-bonding effect involves secondary bridging hydrogen bonding from the primary water-solvation shell of the transition state and the growth of structured water clusters. Theoretical calculations strongly support these conclusions.