Improved protocol for histological and histopathological preparation of large eyes.

The development of new treatments for ocular diseases often requires investigating eyes similar in size and structure to human eyes. Such studies are challenging because analyzing the histopathology of large, human-sized eyes can be technically difficult. In particular, obtaining high-quality frozen sections is almost impossible due to the formation of ice crystals in the vitreous, which causes crush artifacts during the procedures of section and post sectioning manipulations. Herein, we describe a new method that provides high-quality frozen sections for large eyes and demonstrate its usefulness in the eyes of rabbits, pigs, minipigs, monkeys, and humans. We observed that artifactual separation of the photoreceptors from the retinal pigment epithelium is minimized and photoreceptor morphology is preserved. This method can be highly beneficial for investigators seeking to translate new treatments for ocular disease into the clinic. RESEARCH HIGHLIGHTS: Histopathological analysis of large and human-sized eyes presents significant challenges, particularly in obtaining high-quality frozen sections. A multistep fixation followed by vitreous removal and replacement ensures better cryopreservation and embedding of large eyes, minimizing the morphological and structural retinal loss found in many studies. Our results demonstrate that a multistep fixation and cryopreservation method for large eyes in histopathology consistently minimizes the artifactual separation of photoreceptors from the retinal pigment epithelium, thereby preserving photoreceptor morphology and providing high-quality frozen sections. A new method providing high-quality sections is necessary and will be highly useful for investigators aiming to translate new treatments for ocular diseases into clinical applications.

Microbial reaction rate estimation using proteins and proteomes.

Microbes transform their environments using diverse enzymatic reactions. However, it remains challenging to measure microbial reaction rates in natural environments. Despite advances in global quantification of enzyme abundances, the individual relationships between enzyme abundances and their reaction rates have not been systematically examined. Using matched proteomic and reaction rate data from microbial cultures, we show that enzyme abundance is often insufficient to predict its corresponding reaction rate. However, we discovered that global proteomic measurements can be used to make accurate rate predictions of individual reaction rates (median R 2 = 0.78). Accurate rate predictions required only a small number of proteins and they did not need explicit prior mechanistic knowledge or environmental context. These results indicate that proteomes are encoders of cellular reaction rates, potentially enabling proteomic measurements in situ to estimate the rates of microbially mediated reactions in natural systems.

Automated preparation of plasma lipids, metabolites, and proteins for LC/MS-based analysis of a high-fat diet in mice.

Blood plasma is one of the most commonly analyzed and easily accessible biological samples. Here, we describe an automated liquid-liquid extraction platform that generates accurate, precise, and reproducible samples for metabolomic, lipidomic, and proteomic analyses from a single aliquot of plasma while minimizing hands-on time and avoiding contamination from plasticware. We applied mass spectrometry to examine the metabolome, lipidome, and proteome of 90 plasma samples to determine the effects of age, time of day, and a high-fat diet in mice. From 25 µl of mouse plasma, we identified 907 lipid species from 16 different lipid classes and subclasses, 233 polar metabolites, and 344 proteins. We found that the high-fat diet induced only mild changes in the polar metabolome, upregulated apolipoproteins, and induced substantial shifts in the lipidome, including a significant increase in arachidonic acid and a decrease in eicosapentaenoic acid content across all lipid classes.

Suprachoroidal gene transfer with nonviral nanoparticles in large animal eyes.

Suprachoroidal nonviral gene therapy with biodegradable poly(ß-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented epithelial (RPE) cells and therapeutic benefits in rodents. Here, we show in a human-sized minipig eye that suprachoroidal injection of 50 µl of NPs containing 19.2 µg of GFP expression plasmid caused GFP expression in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after injection of 50, 100, or 200 µl, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 µl and markedly reduced after three suprachoroidal injections at different locations around the eye. Reduction of bacterial CpG sequences in the expression plasmid resulted in a trend toward higher expression. These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-sized eye.

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

Reduced inspired oxygen decreases retinal superoxide radicals and promotes cone function and survival in a model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by many different mutations that promote the degeneration of rod photoreceptors and have no direct effect on cones. After the majority of rods have died cone photoreceptors begin to slowly degenerate. Oxidative damage contributes to cone cell death and it has been hypothesized that tissue hyperoxia due to reduced oxygen consumption from the loss of rods is what initiates oxidative stress. Herein, we demonstrate in animal models of RP that reduction of retinal hyperoxia by reducing inspired oxygen to continuous breathing of 11% O2 reduced the generation of superoxide radicals in the retina and preserved cone structure and function. These data indicate that retinal hyperoxia is the initiating event that promotes oxidative damage, loss of cone function, and cone degeneration in the RP retina.

Oxidative stress-induced alterations in retinal glucose metabolism in Retinitis Pigmentosa.

Retinitis pigmentosa occurs due to mutations that cause rod photoreceptor degeneration. Once most rods are lost, gradual degeneration of cone photoreceptors occurs. Oxidative damage and abnormal glucose metabolism have been implicated as contributors to cone photoreceptor death. Herein, we show increased phosphorylation of key enzymes of glucose metabolism in the retinas of rd10 mice, a model of RP, and retinas of wild type mice with paraquat-induced oxidative stress, thereby inhibiting these key enzymes. Dietary supplementation with glucose and pyruvate failed to overcome the inhibition, but increased reducing equivalents in the retina and improved cone function and survival. Dichloroacetate reversed the increased phosphorylation of pyruvate dehydrogenase in rd10 retina and increased histone acetylation and levels of TP53-induced glycolysis and apoptosis regulator (TIGAR), which redirected glucose metabolism toward the pentose phosphate pathway. These data indicate that oxidative stress induced damage can be reversed by shifting glycolytic intermediates toward the pentose phosphate pathway which increases reducing equivalents and provides photoreceptor protection.

Evaluation of Williams et al.: On multiomics and mediation.

One snapshot of the peer review process for "Multiomic profiling of the liver across diets and age in a diverse mouse population" (Williams et al., 2021).

Sugar phosphate activation of the stress sensor eIF2B.

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

Proteosomal degradation impairs transcytosis of AAV vectors from suprachoroidal space to retina.

Suprachoroidal injection provides a new route of delivery for AAV vectors to retinal pigmented epithelial cells and photoreceptors that can be done in an outpatient setting and is less invasive and potentially safer than subretinal injection, the most common route of delivery for ocular gene therapy. After suprachoroidal injection of AAV8 or AAV9 vectors, there is strong transduction of photoreceptors, but it is unclear how vector traverses the retinal pigmented epithelium. In this study, we found that transduction of photoreceptors was significantly increased after suprachoroidal injection of AAV2tYF-CBA-GFP versus AAV2-CBA-GFP vector. Compared with AAV2, AAV2tYF is more resistant to proteosomal degradation. Treatment with protease inhibitors significantly increased photoreceptor transduction after suprachoroidal injection of AAV5-GRK1-GFP. These data suggest that after suprachoroidal injection, AAV vectors access photoreceptors by transcytosis through retinal pigmented epithelial cells during which they are subject to proteosomal degradation, which if suppressed can enhance transduction of photoreceptors.

Gelling hypotonic polymer solution for extended topical drug delivery to the eye.

Eye-drop formulations should hold as high a concentration of soluble drug in contact with ocular epithelium for as long as possible. However, eye tears and frequent blinking limit drug retention on the ocular surface, and gelling drops typically form clumps that blur vision. Here, we describe a gelling hypotonic solution containing a low concentration of a thermosensitive triblock copolymer for extended ocular drug delivery. On topical application, the hypotonic formulation forms a highly uniform and clear thin layer that conforms to the ocular surface and resists clearance from blinking, increasing the intraocular absorption of hydrophilic and hydrophobic drugs and extending the drug-ocular-epithelium contact time with respect to conventional thermosensitive gelling formulations and commercial eye drops. We also show that the conformal gel layer allows for therapeutically relevant drug delivery to the posterior segment of the eyeball in pigs. Our findings highlight the importance of formulations that conform to the ocular surface before viscosity enhancement for increased and prolonged ocular surface contact and drug absorption.

Hepatocyte growth factor is upregulated in ischemic retina and contributes to retinal vascular leakage and neovascularization.

In patients with macular edema due to ischemic retinopathy, aqueous levels of hepatocyte growth factor (HGF) correlate with edema severity. We tested whether HGF expression and activity in mice with oxygen-induced ischemic retinopathy supports a role in macular edema. In ischemic retina, HGF was increased in endogenous cells and macrophages associated with retinal neovascularization (NV). HGF activator was increased in and around retinal vessels potentially providing vascular targeting. One day after intravitreous injection of HGF, VE-cadherin was reduced and albumin levels in retina and vitreous were significantly increased indicating vascular leakage. Injection of VEGF caused higher levels of vitreous albumin than HGF, and co-injection of both growth factors caused significantly higher levels than either alone. HGF increased the number of macrophages on the retinal surface, which was blocked by anti-c-Met and abrogated in chemokine (C-C motif) ligand 2 (CCL2)-/- mice. Injection of anti-c-Met significantly decreased leakage within 24 hours and after 5 days it reduced retinal NV in mice with ischemic retinopathy, but had no effect on choroidal NV. These data indicate that HGF is a pro-permeability, pro-inflammatory, and pro-angiogenic factor and along with its activator is increased in ischemic retina providing support for a potential role of HGF in macular edema in ischemic retinopathies.

Learning causal networks using inducible transcription factors and transcriptome-wide time series.

We present IDEA (the Induction Dynamics gene Expression Atlas), a dataset constructed by independently inducing hundreds of transcription factors (TFs) and measuring timecourses of the resulting gene expression responses in budding yeast. Each experiment captures a regulatory cascade connecting a single induced regulator to the genes it causally regulates. We discuss the regulatory cascade of a single TF, Aft1, in detail; however, IDEA contains > 200 TF induction experiments with 20 million individual observations and 100,000 signal-containing dynamic responses. As an application of IDEA, we integrate all timecourses into a whole-cell transcriptional model, which is used to predict and validate multiple new and underappreciated transcriptional regulators. We also find that the magnitudes of coefficients in this model are predictive of genetic interaction profile similarities. In addition to being a resource for exploring regulatory connectivity between TFs and their target genes, our modeling approach shows that combining rapid perturbations of individual genes with genome-scale time-series measurements is an effective strategy for elucidating gene regulatory networks.

Sustained delivery of acriflavine from the suprachoroidal space provides long term suppression of choroidal neovascularization.

Hypoxia-inducible factor-1 (HIF-1) has been implicated in the pathogenesis of choroidal neovascularization (NV) and is an appealing target because it increases multiple pro-angiogenic proteins and their receptors. Acriflavine (ACF) binds HIF-1α and HIF-2α preventing binding to HIF-1ß and inhibiting transcriptional activity of HIF-1 and HIF-2. Delivery of ACF to the eye by multiple routes strongly, but transiently, suppresses choroidal NV. We overcame design challenges and loaded highly water soluble ACF into poly(lactic-co-glycolic acid) (PLGA) microparticles (PLGA-ACF MPs) that release ACF in vitro for up to 60 days. Intravitreous injection of PLGA-ACF MPs in mice suppressed choroidal NV for at least 9 weeks and suprachoroidal injection of PLGA-ACF in rats suppressed choroidal NV for at least 18 weeks. Intravitreous, but not suprachoroidal injection, of PLGA-ACF MPs containing 38 µg of ACF in rabbits resulted in modest reduction of full-field electroretinogram (ERG) function. Over the span of 28 days after suprachoroidal injection of PLGA-ACF MP, rabbits had normal appearing retinas on fundus photographs, normal electroretinogram scotopic a- and b-wave amplitudes, no increase in intraocular pressure, and normal retinal histology. The active component of ACF, trypaflavine, had steady-state levels in the low nM range in RPE/choroid > retina for at least 16 weeks with a gradient from the side of the eye where the injection was done to the opposite side. These data suggest that suprachoroidal injection of PLGA-ACF MPs has the potential to provide a durable new treatment for retinal and choroidal vascular diseases.

Impact of a Pharmacy-Initiated Inpatient Diabetes Patient Education Program on 30-Day Readmission Rates.

BACKGROUND: In October 2012, a pharmacy-driven Inpatient Diabetes Patient Education (IDPE) program was implemented at the University of Toledo Medical Center (UTMC). OBJECTIVE: To determine the difference in 30-day hospital readmission rates for patients who receive IDPE compared to those who do not. METHODS: This retrospective cohort was completed at UTMC. Patients admitted between October 1, 2012, and September 30, 2013, were included if they were ≥18 years and had one of the following: (1) diagnosis of diabetes mellitus, (2) blood glucose >200 mg/dL (>11.11 mmol/L) on admission, or (3) hemoglobin A1C of >6.5% (>48 mmol/mol). Patients who received IDPE from a pharmacist or student pharmacist (intervention group) were compared to patients who did not receive IDPE (control group). RESULTS: The 30-day readmission rate was 13.2% for the intervention group (n = 364) and 21.5% for the control group (n = 149) (P = .023). Average time to 30-day readmission was 13.1 (±8.3) days for the IDPE group and 11.9 (±7.9) days for the control group. There was no significant difference in diabetes-related readmission between the intervention and control groups (25.5% vs 21.9%). CONCLUSIONS: An IDPE program delivered primarily by pharmacists and student pharmacists significantly reduced 30-day readmission rates among patients with diabetes.

Comprehensive analysis of a mouse model of spontaneous uveoretinitis using single-cell RNA sequencing.

Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire-/- mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire-/- retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire-/- retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire-/- retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.

An innovative non-invasive technique for subcutaneous tumour measurements.

INTRODUCTION: In oncological drug development, animal studies continue to play a central role in which the volume of subcutaneous tumours is monitored to assess the efficacy of new drugs. The tumour volume is estimated by taking the volume to be that of a regular spheroid with the same dimensions. However, this method is subjective, insufficiently traceable, and is subject to error in the accuracy of volume estimates as tumours are frequently irregular. METHODS & RESULTS: This paper reviews the standard technique for tumour volume assessment, calliper measurements, by conducting a statistical review of a large dataset consisting of 2,500 tumour volume measurements from 1,600 mice by multiple operators across 6 mouse strains and 20 tumour models. Additionally, we explore the impact of six different tumour morphologies on volume estimation and the detection of treatment effects using a computational tumour growth model. Finally, we propose an alternative method to callipers for estimating volume-BioVolumeTM, a 3D scanning technique. BioVolume simultaneously captures both stereo RGB (Red, Green and Blue) images from different light sources and infrared thermal images of the tumour in under a second. It then detects the tumour region automatically and estimates the tumour volume in under a minute. Furthermore, images can be processed in parallel within the cloud and so the time required to process multiple images is similar to that required for a single image. We present data of a pre-production unit test consisting of 297 scans from over 120 mice collected by four different operators. CONCLUSION: This work demonstrates that it is possible to record tumour measurements in a rapid minimally invasive, morphology-independent way, and with less human-bias compared to callipers, whilst also improving data traceability. Furthermore, the images collected by BioVolume may be useful, for example, as a source of biomarkers for animal welfare and secondary drug toxicity / efficacy.

AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression.

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

Insect pest control, approximate dynamic programming, and the management of the evolution of resistance.

Ecological decision problems, such as those encountered in agriculture, often require managing conflicts between short-term costs and long-term benefits. Dynamic programming is an ideal method for optimally solving such problems but agricultural problems are often subject to additional complexities that produce state spaces intractable to exact solutions. In contrast, look-ahead policies, a class of approximate dynamic programming (ADP) algorithm, may attempt to solve problems of arbitrary magnitude. However, these algorithms focus on a temporally truncated caricature of the full decision problem over a defined planning horizon and as such are not guaranteed to suggest optimal actions. Thus, look-ahead policies may offer promising means of addressing detail-rich ecological decision problems but may not be capable of fully utilizing the information available to them, especially in scenarios where the best short- and long-term solutions may differ. We constructed and applied look-ahead policies to the management of a hypothetical, stage-structured, continually reproducing, agricultural insect pest. The management objective was to minimize the combined costs of management actions and crop damage over a 16-week growing season. The manager could elect to utilize insecticidal sprays or one of six release ratios of male-selecting transgenic insects where the release ratio determines the number of transgenic insects to be released for each wild-type male insect in the population. Complicating matters was the expression of insecticide resistance at non-trivial frequencies in the pest population. We assessed the extent to which look-ahead policies were able to recognize the potential threat of insecticide resistance and successfully integrate insecticides and transgenic releases to capitalize upon their respective benefits. Look-ahead policies were competent at anticipating and responding to ecological and economic information. Policies with longer planning horizons made fewer, better-timed insecticidal sprays and made more frequent transgenic releases, which consequently facilitated lower resistance allele frequencies. However, look-ahead policies were ultimately inefficient resistance managers, and directly responded to resistance only when it was dominant and prevalent. Effective long-term agricultural management requires the capacity to anticipate and respond to the evolution of resistance. Look-ahead policies can accommodate all the information pertinent to making the best long-term decision but may lack the perspective to actually do so.

Identification of novel transcripts and peptides in developing murine lens.

We previously investigated the transcriptome and proteome profiles of the murine ocular lens at six developmental time points including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). Here, we extend our analyses to identify novel transcripts and peptides in developing  mouse lens. We identified a total of 9,707 novel transcripts and 325 novel fusion genes in developing mouse lens. Additionally, we identified 13,281 novel alternative splicing (AS) events in mouse lens including 6,990 exon skipping (ES), 2,447 alternative 3' splice site (A3SS), 1,900 alternative 5' splice site (A5SS), 1,771 mutually exclusive exons (MXE), and 173 intron retention (IR). Finally, we integrated our OMIC (Transcriptome and Proteome) datasets identifying 20 novel peptides in mouse lens. All 20 peptides were validated through matching MS/MS spectra of synthetic peptides. To the best of our knowledge, this is the first report integrating OMIC datasets to identify novel peptides in developing murine lens.