Addressing Technical Failures in a Diabetic Retinopathy Screening Program.

Purpose: Diabetic retinopathy (DR) is a preventable cause of blindness detectable through screening using retinal digital photography. The Irish National Diabetic Retina Screening (DRS) programme, Diabetic RetinaScreen, provides free screening services to patients with diabetes from aged 12 years and older. A technical failure (TF) occurs when digital retinal imaging is ungradable, resulting in delays in the diagnosis and treatment of sight-threatening disease. Despite their impact, the causes of TFs, and indeed the utility of interventions to prevent them, have not been extensively examined. Aim: Primary analysis aimed to identify factors associated with TF. Secondary analysis examined a subset of cases, assessing patient data from five time points between 2019 and 2021 to identify photographer/patient factors associated with TF. Methods: Patient data from the DRS database for one provider were extracted for analysis between 2018 and 2022. Information on patient demographics, screening results, and other factors previously associated with TF were analyzed. Primary analysis involved using mixed-effects logistic regression models with nested patient-eye random effects. Secondary analysis reviewed a subset of cases in detail, checking for causes of TF. Results: The primary analysis included a total of 366,528 appointments from 104,407 patients over 5 years. Most patients had Type 2 diabetes (89.2%), and the overall TF rate was 4.9%. Diabetes type and duration, dilate pupil status, and the presence of lens artefacts on the camera were significantly associated with TF. The Secondary analysis identified the primary cause of TF was found to be optically dense cataracts, accounting for over half of the TFs. Conclusion: This study provides insight into the causes of TF within the Irish DRS program, highlighting cataracts as the primary contributing factor. The identification of patient-level factors associated with TF facilitates appropriate interventions that can be put in place to improve patient outcomes and minimize delays in treatment and diagnosis.

Single Live Cell Imaging of Multidrug Resistance Using Silver Ultrasmall Nanoparticles as Biosensing Probes in Triple-Negative Breast Cancer Cells.

Silver ultrasmall nanoparticles (Ag UNPs) (size < 5 nm) were used as biosensing probes to analyze the efflux kinetics contributing to multidrug resistance (MDR) in single live triple-negative breast cancer (TNBC) cells by using dark-field optical microscopy to follow their size-dependent localized surface plasmon resonance. TNBC cells lack expression of estrogen (ER-), progesterone (PR-), and human epidermal growth factor 2 (HER2-) receptors and are more likely to acquire resistance to anticancer drugs due to their ability to transport harmful substances outside the cell. The TNBC cells displayed greater nuclear and cytoplasmic efflux, resulting in less toxicity of Ag UNPs in a concentration-independent manner. In contrast, more Ag UNPs and an increase in cytotoxic effects were observed in the receptor-positive breast cancer cells that have receptors for ER+, PR+, and HER2+ and are known to better respond to anticancer therapies. Ag UNPs accumulated in receptor-positive breast cancer cells in a time-and concentration-dependent mode and caused decreased cellular growth, whereas the TNBC cells due to the efflux were able to continue to grow. The TNBC cells demonstrated a marked increase in survival due to their ability to have MDR determined by efflux of Ag UNPs outside the nucleus and the cytoplasm of the cells. Further evaluation of the nuclear efflux kinetics of TNBC cells with Ag UNPs as biosensing probes is critical to gain a better understanding of MDR and potential for enhancement of cancer drug delivery.

Comparing confocal and two-photon Ca2+ imaging of thin low-scattering preparations.

Ca2+ imaging provides insight into biological processes ranging from subcellular dynamics to neural network activity. Two-photon microscopy has assumed a dominant role in Ca2+ imaging. The longer wavelength infra-red illumination undergoes less scattering, and absorption is confined to the focal plane. Two-photon imaging can thus penetrate thick tissue ∼10-fold more deeply than single-photon visible imaging to make two-photon microscopy an exceptionally powerful method for probing function in intact brain. However, two-photon excitation produces photobleaching and photodamage that increase very steeply with light intensity, limiting how strongly one can illuminate. In thin samples, illumination intensity can assume a dominant role in determining signal quality, raising the possibility that single-photon microscopy may be preferable. We therefore tested laser scanning single-photon and two-photon microscopy side by side with Ca2+ imaging in neuronal compartments at the surface of a brain slice. We optimized illumination intensity for each light source to obtain the brightest signal without photobleaching. Intracellular Ca2+ rises elicited by one action potential had twice the signal/noise ratio with confocal as with two-photon imaging in axons, were 31% higher in dendrites, and about the same in cell bodies. The superior performance of confocal imaging in finer neuronal processes likely reflects the dominance of shot noise when fluorescence is dim. Thus, when out-of-focus absorption and scattering are not issues, single-photon confocal imaging can yield better quality signals than two-photon microscopy.

The Effect on Patients' Visual Acuity and Grade, Secondary to Non-Attendance at Treatment Centers, Post Referral from Diabetic RetinaScreen Ireland.

Background: Patient non-attendance following referral to hospital is a significant challenge, in particular, for persons with diabetes. Aim: We sought to determine the impact on both visual acuity and the subsequent follow-up retinopathy grade of patients when they fail to attend Diabetic Retinopathy Treatment (DRT) Centers following referral from Diabetic RetinaScreen (DRS). Methods: A retrospective analysis of patients discharged from DRT due to multiple consecutive missed appointments between January 2016 and June 2021. Patients discharged for non-attendance were compared with patients discharged from completed treatment. Results: Of the 24,945 NEC patients referred to DRT, 5900 (24%) and 9345 (37%) were discharged back to DRS due to non-attendance and completed treatment, respectively. Those discharged for non-attendance were younger (60.7 v 63.4, p < 0.001) and had higher proportions of males (67% v 63%, p < 0.001) and people with type 1 diabetes (27% v 18%, p < 0.001). After attending rescreening after discharge, those discharged for non-attendance were significantly more likely to have a worsening of DR grade (26% v 8%, p < 0.001). Conclusion: Despite being notified that further investigation (with possible treatment) was required post DRS, many diabetic patients failed to attend for further management of their eye care in DRT. These patients had worse visual outcomes compared to those that attended. Improved patient education and communication are required to mitigate against the consequences of non-attendance.

Direct synaptic excitation between hilar mossy cells revealed with a targeted voltage sensor.

The dentate gyrus not only gates the flow of information into the hippocampus, it also integrates and processes this information. Mossy cells (MCs) are a major type of excitatory neuron strategically located in the hilus of the dentate gyrus where they can contribute to this processing through networks of synapses with inhibitory neurons and dentate granule cells. Some prior work has suggested that MCs can form excitatory synapses with other MCs, but the role of these synapses in the network activity of the dentate gyrus has received little attention. Here, we investigated synaptic inputs to MCs in mouse hippocampal slices using a genetically encoded hybrid voltage sensor (hVOS) targeted to MCs by Cre-lox technology. This enabled optical recording of voltage changes from multiple MCs simultaneously. Stimulating granule cells and CA3 pyramidal cells activated well-established inputs to MCs and elicited synaptic responses as expected. However, the weak blockade of MC responses to granule cell layer stimulation by DCG-IV raised the possibility of another source of excitation. To evaluate synapses between MCs as this source, single MCs were stimulated focally. Stimulation of one MC above its action potential threshold evoked depolarizing responses in neighboring MCs that depended on glutamate receptors. Short latency responses of MCs to other MCs did not depend on release from granule cell axons. However, granule cells did contribute to the longer latency responses of MCs to stimulation of other MCs. Thus, MCs transmit their activity to other MCs both through direct synaptic coupling and through polysynaptic coupling with dentate granule cells. MC-MC synapses can redistribute information entering the dentate gyrus and thus shape and modulate the electrical activity underlying hippocampal functions such as navigation and memory, as well as excessive excitation during seizures.

Pembrolizumab-Induced Diabetes Mellitus Presenting as Diabetic Ketoacidosis in a Patient With Metastatic Colonic Adenocarcinoma.

Immunotherapy drugs are gaining popularity in the treatment of certain malignancies due to the success of these agents in recent clinical trials. Pembrolizumab is an immune checkpoint inhibitor that acts via binding to programmed cell death 1 (PD-1) receptors on T-cells, allowing for the constitutive activation of T-cells to fight malignant tumor cells. Immune checkpoint molecules such as PD-1 act to inhibit T-cell function, promoting tolerance to self-antigens. Inhibition of these molecules may lead to increased T-cell activation against cancer cells, but also against healthy tissue, leading to the side effects of these medications known as immune-related adverse events. In this article, we present the case of a 77-year-old female with a history of metastatic colonic adenocarcinoma presenting with new-onset diabetes mellitus and diabetic ketoacidosis in the setting of receiving pembrolizumab chemotherapy. Our patient was treated with hydration, insulin therapy, and management of her electrolytes, ultimately being discharged with the need for home insulin therapy to manage her new-onset diabetes. There are no current guidelines for the management or surveillance of patients receiving pembrolizumab chemotherapy, and further research should be done to determine which patients are at highest risk to developing this rare but potentially lethal side effect.

An Inconvenient Truth: Calcium Sensors Are Calcium Buffers.

Recent advances in Ca2+ imaging have given neuroscientists a tool to follow the activity of large numbers of individual neurons simultaneously in vivo in the brains of animals as they are presented with sensory stimulation, respond to environmental challenges, and engage in behaviors. The Ca2+ sensors used to transduce changes in cellular Ca2+ into changes in fluorescence must bind Ca2+ to produce a signal. By binding Ca2+, these sensors can act as buffers, often reducing the magnitude of a Ca2+ change severalfold, and producing a proportional slowing of the rates of change. Ca2+ probes can thus distort the patterns of activity they are intended to study and modify ongoing Ca2+ signaling functions. Recognizing these factors will enhance the use of in vivo Ca2+ imaging in the investigation of neural circuit function.

Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population.

The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter. There are many avenues for how CRISPR/Cas9 technologies will be applied in vivo, including the mode of delivery. These may be expected to invoke different immunological pathways. Nonetheless, as with all protein therapeutics, it may be desirable to monitor for anti-Cas9 antibodies during clinical development. This will require the development of robust and reliable assays. Here, we describe ELISA-based assays that are capable of detecting antibodies to Cas9 from Staphylococcus aureus (SaCas9) and Streptococcs pyogenes (SpCas9) in human sera. Furthermore, using these assays to screen for pre-existing antibodies in 200 human serum samples, we found the prevalence of anti-SaCas9 and anti-SpCas9 antibodies to be 10% and 2.5%, respectively.

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.

Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.

In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding.

Imaging with Ca(2+)-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca(2+) signaling. The spatiotemporal evolution of intracellular free Ca(2+) observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca(2+) buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca(2+) imaging data, and can be exploited to investigate endogenous Ca(2+) buffer properties. Here we extended the use of Ca(2+) fluorometry in the characterization of Ca(2+) binding molecules within cells, building on a method of titration of intracellular Ca(2+) binding sites in situ with measured amounts of Ca(2+) entering through voltage-gated Ca(2+) channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca(2+) binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca(2+) indicator fluo-8. Focusing on data sets well described by a single endogenous Ca(2+) buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca(2+) inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca(2+)-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca(2+) titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca(2+) binding species. This method offers a versatile approach to the study of endogenous Ca(2+) binding molecules in their physiological milieu.

Excitatory synaptic feedback from the motor layer to the sensory layers of the superior colliculus.

Neural circuits that translate sensory information into motor commands are organized in a feedforward manner converting sensory information into motor output. The superior colliculus (SC) follows this pattern as it plays a role in converting visual information from the retina and visual cortex into motor commands for rapid eye movements (saccades). Feedback from movement to sensory regions is hypothesized to play critical roles in attention, visual image stability, and saccadic suppression, but in contrast to feedforward pathways, motor feedback to sensory regions has received much less attention. The present study used voltage imaging and patch-clamp recording in slices of rat SC to test the hypothesis of an excitatory synaptic pathway from the motor layers of the SC back to the sensory superficial layers. Voltage imaging revealed an extensive depolarization of the superficial layers evoked by electrical stimulation of the motor layers. A pharmacologically isolated excitatory synaptic potential in the superficial layers depended on stimulus strength in the motor layers in a manner consistent with orthodromic excitation. Patch-clamp recording from neurons in the sensory layers revealed excitatory synaptic potentials in response to glutamate application in the motor layers. The location, size, and morphology of responsive neurons indicated they were likely to be narrow-field vertical cells. This excitatory projection from motor to sensory layers adds an important element to the circuitry of the SC and reveals a novel feedback pathway that could play a role in enhancing sensory responses to attended targets as well as visual image stabilization.

Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice.

Gene targeting with genetically encoded optical voltage sensors brings the methods of voltage imaging to genetically defined neurons and offers a method of studying circuit activity in these selected populations. The present study reports the targeting of genetically encoded hybrid voltage sensors (hVOS) to neurons in transgenic mice. The hVOS family of probes employs a membrane-targeted fluorescent protein, which generates voltage-dependent fluorescence changes in the presence of dipicrylamine (DPA) as the result of a voltage-dependent optical interaction between the two molecules. We generated transgenic mice with two different high-performance hVOS probes under control of a neuron-specific thy-1 promoter. Hippocampal slices from these animals present distinct spatial patterns of expression, and electrical stimulation evoked fluorescence changes as high as 3%. Glutamate receptor and Na(+) channel antagonists blocked these responses. One hVOS probe tested here harbors an axonal targeting motif (from GAP-43) and shows preferential expression in axons; this probe can thus report axonal voltage changes. Voltage imaging in transgenic mice expressing hVOS probes opens the door to the study of functional activity in genetically defined populations of neurons in intact neural circuits.