Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Repeated LPS induces training and tolerance of microglial responses across brain regions.

BACKGROUND: Neuroinflammation is involved in the pathogenesis of almost every central nervous system disorder. As the brain's innate immune cells, microglia fine tune their activity to a dynamic brain environment. Previous studies have shown that repeated bouts of peripheral inflammation can trigger long-term changes in microglial gene expression and function, a form of innate immune memory. METHODS AND RESULTS: In this study, we used multiple low-dose lipopolysaccharide (LPS) injections in adult mice to study the acute cytokine, transcriptomic, and microglia morphological changes that contribute to the formation of immune memory in the frontal cortex, hippocampus, and striatum, as well as the long-term effects of these changes on behavior. Training and tolerance of gene expression was shared across regions, and we identified 3 unique clusters of DEGs (2xLPS-sensitive, 4xLPS-sensitive, LPS-decreased) enriched for different biological functions. 2xLPS-sensitive DEG promoters were enriched for binding sites for IRF and NFkB family transcription factors, two key regulators of innate immune memory. We quantified shifts in microglia morphological populations and found that while the proportion of ramified and rod-like microglia mostly remained consistent within brain regions and sexes with LPS treatment, there was a shift from ameboid towards hypertrophic morphological states across immune memory states and a dynamic emergence and resolution of events of microglia aligning end-to-end with repeated LPS. CONCLUSIONS: Together, findings support the dynamic regulation of microglia during the formation of immune memories in the brain and support future work to exploit this model in brain disease contexts.

Ultrasound Imaging of Various Ingested Foreign Bodies in an Ex Vivo Intestinal Model.

OBJECTIVE: Foreign body ingestion is an increasingly prevalent issue for children who are in the preverbal to early verbal stages of life. Foreign bodies lodged in the gastrointestinal tract can cause issues such as obstruction, perforation, and fistulae. Radiographic imaging can often locate most foreign bodies; however, radiolucent objects may be missed. Ultrasound is an alternative imaging modality that can be used to locate and track foreign objects as they pass through the bowel. The objective of this study was to characterize the sonographic appearance of various ingested foreign bodies of varying characteristics in an ex vivo gastrointestinal tract segment. METHODS: A GE Logiq 9 ultrasound machine with a linear transducer at a frequency of 15 MHz was used to examine various ingested foreign bodies placed in a segment of pig intestinal tract. RESULTS: Imaged objects varied in visual appearance from echogenicity, texture, size, and shape; acoustic shadows and reverberation artifacts cast were particularly distinguishing characteristics. CONCLUSIONS: Ultrasound evaluation to assess foreign body ingestion in the pediatric population may provide a useful alternative or supportive imaging modality in confirming the location and real-time tracking of the ingested item. This may be especially useful for objects of varying radiodensities that cannot always be reliably seen in traditional radiographs.

Improved transcriptome assembly using a hybrid of long and short reads with StringTie.

Short-read RNA sequencing and long-read RNA sequencing each have their strengths and weaknesses for transcriptome assembly. While short reads are highly accurate, they are rarely able to span multiple exons. Long-read technology can capture full-length transcripts, but its relatively high error rate often leads to mis-identified splice sites. Here we present a new release of StringTie that performs hybrid-read assembly. By taking advantage of the strengths of both long and short reads, hybrid-read assembly with StringTie is more accurate than long-read only or short-read only assembly, and on some datasets it can more than double the number of correctly assembled transcripts, while obtaining substantially higher precision than the long-read data assembly alone. Here we demonstrate the improved accuracy on simulated data and real data from Arabidopsis thaliana, Mus musculus, and human. We also show that hybrid-read assembly is more accurate than correcting long reads prior to assembly while also being substantially faster. StringTie is freely available as open source software at https://github.com/gpertea/stringtie.

The steroidal lactone withaferin A impedes T-cell motility by inhibiting the kinase ZAP70 and subsequent kinome signaling.

The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 µM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies.

Cardiovascular magnetic resonance predictors of heart failure in hypertrophic cardiomyopathy: the role of myocardial replacement fibrosis and the microcirculation.

INTRODUCTION: Heart failure (HF) in hypertrophic cardiomyopathy (HCM) is associated with high morbidity and mortality. Predictors of HF, in particular the role of myocardial fibrosis and microvascular ischemia remain unclear. We assessed the predictive value of cardiovascular magnetic resonance (CMR) for development of HF in HCM in an observational cohort study. METHODS: Serial patients with HCM underwent CMR, including adenosine first-pass perfusion, left atrial (LA) and left ventricular (LV) volumes indexed to body surface area (i) and late gadolinium enhancement (%LGE- as a % of total myocardial mass). We used a composite endpoint of HF death, cardiac transplantation, and progression to NYHA class III/IV. RESULTS: A total of 543 patients with HCM underwent CMR, of whom 94 met the composite endpoint at baseline. The remaining 449 patients were followed for a median of 5.6 years. Thirty nine patients (8.7%) reached the composite endpoint of HF death (n = 7), cardiac transplantation (n = 2) and progression to NYHA class III/IV (n = 20). The annual incidence of HF was 2.0 per 100 person-years, 95% CI (1.6-2.6). Age, previous non-sustained ventricular tachycardia, LV end-systolic volume indexed to body surface area (LVESVI), LA volume index ; LV ejection fraction, %LGE and presence of mitral regurgitation were significant univariable predictors of HF, with LVESVI (Hazard ratio (HR) 1.44, 95% confidence interval (95% CI) 1.16-1.78, p = 0.001), %LGE per 10% (HR 1.44, 95%CI 1.14-1.82, p = 0.002) age (HR 1.37, 95% CI 1.06-1.77, p = 0.02) and mitral regurgitation (HR 2.6, p = 0.02) remaining independently predictive on multivariable analysis. The presence or extent of inducible perfusion defect assessed using a visual score did not predict outcome (p = 0.16, p = 0.27 respectively). DISCUSSION: The annual incidence of HF in a contemporary ambulatory HCM population undergoing CMR is low. Myocardial fibrosis and LVESVI are strongly predictive of future HF, however CMR visual assessment of myocardial perfusion was not.

Upregulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain.

BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed with western blotting and its cellular localization was determined using immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum, was measured with enzyme-linked immunosorbent assay (ELISA).ResultsThere was a transient cholesterol loss at 6 h after HI. CYP46A1 was significantly upregulated at 6 and 24 h following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6 and 24 h after HI.ConclusionEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.

Shaping Streamflow Using a Real-Time Stormwater Control Network.

"Smart" water systems are transforming the field of stormwater management by enabling real-time monitoring and control of previously static infrastructure. While the localized benefits of active control are well-established, the potential for system-scale control of watersheds is poorly understood. This study shows how a real-world smart stormwater system can be leveraged to shape streamflow within an urban watershed. Specifically, we coordinate releases from two internet-controlled stormwater basins to achieve desired control objectives downstream-such as maintaining the flow at a set-point, and generating interleaved waves. In the first part of the study, we describe the construction of the control network using a low-cost, open-source hardware stack and a cloud-based controller scheduling application. Next, we characterize the system's control capabilities by determining the travel times, decay times, and magnitudes of various waves released from the upstream retention basins. With this characterization in hand, we use the system to generate two desired responses at a critical downstream junction. First, we generate a set-point hydrograph, in which flow is maintained at an approximately constant rate. Next, we generate a series of overlapping and interleaved waves using timed releases from both retention basins. We discuss how these control strategies can be used to stabilize flows, thereby mitigating streambed erosion and reducing contaminant loads into downstream waterbodies.

Smarter Stormwater Systems.

Existing stormwater systems require significant investments to meet challenges imposed by climate change, rapid urbanization, and evolving regulations. There is an unprecedented opportunity to improve urban water quality by equipping stormwater systems with low-cost sensors and controllers. This will transform their operation from static to adaptive, permitting them to be instantly "redesigned" to respond to individual storms and evolving land uses.

Microfabrication of High-Resolution Porous Membranes for Cell Culture.

Microporous membranes are widely utilized in cell biology to study cell-cell signaling and cell migration. However, the thickness and low porosity of commercial track-etched membranes limit the quality of cell imaging and the degree of cell-cell contact that can be achieved on such devices. We employ photolithography-based microfabrication to achieve porous membranes with pore diameter as small as 0.9 µm, up to 40% porosity, and less than 5% variation in pore size. Through the use of a soap release layer, membranes as thin as 1 µm can be achieved. The thin membranes minimally disrupt contrast enhancement optics, thus allowing good quality imaging of unlabeled cells under white light, unlike commercial membranes. In addition, the polymer membrane materials display low autofluorescence even after patterning, facilitating high quality fluorescence microscopy. Finally, confocal imaging suggests that substantial cell-cell contact is possible through the pores of these thin membranes. This membrane technology can enhance existing uses of porous membranes in cell biology as well as enable new types of experiments.

Expert-Level Detection of Referable Glaucoma from Fundus Photographs in a Safety Net Population: The AI and Teleophthalmology in Los Angeles Initiative.

Purpose: To develop and test a deep learning (DL) algorithm for detecting referable glaucoma in the Los Angeles County (LAC) Department of Health Services (DHS) teleretinal screening program. Methods: Fundus photographs and patient-level labels of referable glaucoma (defined as cup-to-disc ratio [CDR] ≥ 0.6) provided by 21 trained optometrist graders were obtained from the LAC DHS teleretinal screening program. A DL algorithm based on the VGG-19 architecture was trained using patient-level labels generalized to images from both eyes. Area under the receiver operating curve (AUC), sensitivity, and specificity were calculated to assess algorithm performance using an independent test set that was also graded by 13 clinicians with one to 15 years of experience. Algorithm performance was tested using reference labels provided by either LAC DHS optometrists or an expert panel of 3 glaucoma specialists. Results: 12,098 images from 5,616 patients (2,086 referable glaucoma, 3,530 non-glaucoma) were used to train the DL algorithm. In this dataset, mean age was 56.8 ± 10.5 years with 54.8% females and 68.2% Latinos, 8.9% Blacks, 2.7% Caucasians, and 6.0% Asians. 1,000 images from 500 patients (250 referable glaucoma, 250 non-glaucoma) with similar demographics (p ≥ 0.57) were used to test the DL algorithm. Algorithm performance matched or exceeded that of all independent clinician graders in detecting patient-level referable glaucoma based on LAC DHS optometrist (AUC = 0.92) or expert panel (AUC = 0.93) reference labels. Clinician grader sensitivity (range: 0.33-0.99) and specificity (range: 0.68-0.98) ranged widely and did not correlate with years of experience (p ≥ 0.49). Algorithm performance (AUC = 0.93) also matched or exceeded the sensitivity (range: 0.78-1.00) and specificity (range: 0.32-0.87) of 6 LAC DHS optometrists in the subsets of the test dataset they graded based on expert panel reference labels. Conclusions: A DL algorithm for detecting referable glaucoma developed using patient-level data provided by trained LAC DHS optometrists approximates or exceeds performance by ophthalmologists and optometrists, who exhibit variable sensitivity and specificity unrelated to experience level. Implementation of this algorithm in screening workflows could help reallocate eye care resources and provide more reproducible and timely glaucoma care.

Maladaptive T-Cell Metabolic Fitness in Autoimmune Diseases.

Immune surveillance and adaptive immune responses, involving continuously circulating and tissue-resident T-lymphocytes, provide host defense against infectious agents and possible malignant transformation while avoiding autoimmune tissue damage. Activation, migration, and deployment of T-cells to affected tissue sites are crucial for mounting an adaptive immune response. An effective adaptive immune defense depends on the ability of T-cells to dynamically reprogram their metabolic requirements in response to environmental cues. Inability of the T-cells to adapt to specific metabolic demands may skew cells to become either hyporesponsive (creating immunocompromised conditions) or hyperactive (causing autoimmune tissue destruction). Here, we review maladaptive T-cell metabolic fitness that can cause autoimmune diseases and discuss how T-cell metabolic programs can potentially be modulated to achieve therapeutic benefits.

Effectiveness of Glaucoma Screening and Factors Associated with Follow-up Adherence among Glaucoma Suspects in a Safety-Net Teleretinal Screening Program.

PURPOSE: To evaluate rates and risk factors associated with follow-up adherence to in-person glaucoma evaluations and confirmed glaucoma diagnosis in glaucoma suspects identified through teleretinal diabetic retinopathy screening (TDRS). DESIGN: Retrospective cohort study SUBJECTS: Patients with diabetes identified through teleretinal screening to have large or asymmetric cup-to-disc ratios in a Los Angeles County safety-net primary care-based TDRS program. METHODS: Retrospective chart review was performed to obtain demographic and clinical information for patients with cup-to-disc ratios concerning for glaucoma on TDRS. Patients who completed an in-person follow-up appointment within 1 year of teleretinal screening were adherent. Factors associated with follow-up adherence and diagnosis of glaucoma were analyzed with chi-square and independent t tests along with multivariable logistic regressions. MAIN OUTCOME MEASURES: The proportion of patients with suspected glaucoma who adhered with in-person follow-up examination, proportion of patients with confirmed glaucoma diagnosis, and factors associated with follow-up adherence and glaucoma diagnosis. RESULTS: Eight-hundred seventeen patients with optic discs suspicious for glaucoma were included. Five-hundred thirty-four (65.4%) patients successfully completed an in-person glaucoma evaluation. Among these patients, 62.9% and 24.5% received a diagnosis of glaucoma suspect and glaucomatous optic neuropathy, respectively. Compared with patients aged < 50 years, patients aged 50 to 64 years had 1.57 times higher odds of being adherent with in-person visits (P = 0.036), whereas no difference was seen in those aged ≥ 65 years. For every $10 000 increase in the zip code median income, patients had 11% lower odds of being adherent (P = 0.031). Compared with Latino patients, Black patients had 3.52 times (P < 0.001) higher odds of having confirmed glaucoma. CONCLUSION: The majority of patients referred as glaucoma suspects on TDRS completed a follow-up examination, and nearly a quarter of those examined received a confirmed glaucoma diagnosis. Patients aged ≥ 50 and < 65 years along with those from lower-income neighborhoods were more likely to follow up for an in-person evaluation. Compared with Latino patients, Black patients had a higher risk for a confirmed glaucoma diagnosis. This demonstrates the effectiveness of glaucoma detection in a large-scale TDRS program for a safety-net patient population. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Long-term evolution of proliferating cells using the eVOLVER platform.

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new stand-alone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled set-up for long-term experimental evolution.

High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.

Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci-especially those with pyrimidine-rich PAM sequences-remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to N4TN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at N4CN PAM sequences.

Rates and Patterns of Diagnostic Conversion from Anatomical Narrow Angle to Primary Angle-Closure Glaucoma in the United States.

PURPOSE: To assess rates of diagnostic conversion from anatomical narrow angle (ANA) to primary angle-closure glaucoma (PACG) in the United States and identify factors associated with diagnostic conversion. DESIGN: Retrospective case-control study. PARTICIPANTS: Patients diagnosed with ANA between the years 2007 and 2019 were identified based on International Classification of Diseases (ICD) codes in the Optum Clinformatics Data Mart Database. Inclusion was limited to newly diagnosed ANA, defined as the following: (1) continuous enrollment during a 2-year look back period and 6-year study period from index (first) date of ANA diagnosis; (2) diagnosis by an ophthalmologist or optometrist and record of gonioscopy; and (3) no history of intraocular pressure (IOP)-lowering drops, laser peripheral iridotomy (LPI), or intraocular surgery. METHODS: Cox proportional hazards models were developed to assess factors associated with diagnostic conversion, defined as a change in ICD code from ANA to PACG. MAIN OUTCOME MEASURES: New diagnosis of PACG within the 6-year study period recorded after an index diagnosis of ANA. RESULTS: Among 3985 patients meeting inclusion criteria, 459 (11.52%) had detected diagnostic conversion to PACG within the study period. The conversion rate was stable at 3.54% per year after the first 6 months of ANA diagnosis. In the Cox proportional hazards model, age > 70 years and early (within 6 months of ANA diagnosis) need for LPI or IOP-lowering drops were positively associated with diagnostic conversion (hazard ratio [HR] > 1.59; P < 0.02). Cataract surgery at any time and late (after 6 months of ANA diagnosis) need for IOP-lowering drops appeared protective against diagnostic conversion (HR < 0.46; P < 0.004). CONCLUSIONS: Annual risk of diagnostic conversion from ANA to PACG is relatively low overall; elderly patients are at higher risk whereas patients receiving cataract surgery are at lower risk. The utility of long-term monitoring seems low for most patients with ANA, highlighting the need for improved clinical methods to identify patients at higher risk for PACG. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Long-term evolution of proliferating cells using the eVOLVER platform.

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently-developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new standalone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled setup for long-term experimental evolution.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Ischemic index and distribution of retinal capillary non-perfusion in neovascular glaucoma.

INTRODUCTION: Neovascular Glaucoma (NVG) is a condition normally caused by hypoxic posterior ocular disease, which produces angiogenic factors such as vascular endothelial growth factor (VEGF) that stimulate new vessel proliferation of the anterior segment and angle, eventually leading to angle closure, reduced outflow of aqueous humor and increased intraocular pressure. Without treatment elevated intraocular pressure can rapidly progress to loss of vision. Treatment includes addressing the intraocular pressure and reducing the ischemic drive with panretinal photocoagulation (PRP) of the ischemic retina. Recent imaging advancements allow for ultra-widefield fluorescein angiography (UWFA) which expand the amount of peripheral retina that can be evaluated for non-perfusion. Here we aim to study patterns of non-perfusion in NVG using a group of PRP naïve patients with recent onset NVG. METHODS: This study is a retrospective single-center cross-sectional study of patients seen at LAC + USC Medical Center from January 2015 to April 2020 with new onset NVG, without PRP and with UWFA completed. The percentage of ischemic index of the retina was calculated from the UWFA and evaluated in three distinct zones centered on the fovea: the posterior pole, the mid periphery, and far periphery. To increase sample size, a confirmatory group was included, with PRP allowed prior to UWFA but not before diagnosis. In addition, the time between diagnosis and UWFA was increased to 6 months. RESULTS: A total of 11 eyes met inclusion criteria for the primary group. Ischemic index was found to be 91% in the far periphery, 77% in the mid periphery, and 42% at the posterior pole. The total average ischemic index was 76%. There was a statistically significant difference between the far periphery and posterior pole and mid periphery and posterior pole. A total of 24 eyes met criteria for the confirmatory group. Ischemic index for the confirmatory group was found to be 93% in the far periphery, 75% in the mid periphery, and 35% at the posterior pole. There was a statistically significant difference between the far periphery, posterior pole and mid-periphery. CONCLUSION: This knowledge can be used to further guide treatment and understand risk for NVG.