Obtaining absorbance spectra from turbid retinal cell and tissue suspensions - Beating the light-scatter problem.

Light scattering and inability to uniformly expose the cuvette contents to an incident light beam are significant limitations of traditional spectrophotometers. The first of these drawbacks limits their usefulness in studies of turbid cellular and tissue suspensions; the second limits their use in photodecomposition studies. Our strategy circumvents both problems. Although we describe its potential usefulness in vision sciences, application of spherical integrating cuvettes has broad application. Absorbance spectra of turbid bovine rod outer segments and dispersed living frog retina were studied using a standard single-pass 1 cm cuvettes, or a spherical integrating cuvette (DeSa Presentation Chamber, DSPC). The DSPC was mounted on an OLIS Rapid Scanning Spectrophotometer configured to generate 100 spectral scans/sec. To follow rhodopsin bleaching kinetics in living photoreceptors, portions of dark-adapted frog retina were suspended in the DSPC. The incoming spectral beam at 2 scans/sec entered the chamber through a single port. Separate ports contained a 519 nm light emitting diode (LED), or window to the photomultiplier tube. The surface of the DSPC was coated with a highly reflective coating allowing the chamber to act as a multi-pass cuvette. The LED is triggered to flash and the PMT shutter temporarily closed during a "Dark-Interval" between each spectral scan. By interleafing scans with LED pulses, spectra changes can be followed in real time. Kinetic analysis of the 3-dimensional data was performed by Singular Value Decomposition. For crude bovine rod outer segment suspensions, the 1 cm single-pass traditional cuvette gave non-informative spectra dominated by high absorbances and Rayleigh scattering. In contrast, spectra generated using the DSPC showed low overall absorbance with peaks at 405 and 503 nm. The later peak disappeared with exposure to white light in presence of 100 mM hydroxylamine. For the dispersed living retinal, the sample was pulsed at 519 nm between the spectra. The 495 nm rhodopsin peak gradually reduced in size concomitant with the emergence of a 400 nm peak, probably representing Meta II. A conversion mechanism of two species, A → B with rate constant of 0.132 sec-1 was fit to the data. To our knowledge this is the first application of integrating sphere technology to retinal spectroscopy. Remarkably, the spherical cuvette designed for total internal reflectance to produce diffused light was efffectively immune to light scattering. Furthermore, the higher effective path length enhanced sensitivity and could be accounted for mathematically allowing determination of absorbance/cm. The approach, which complements the use of the CLARiTy RSM 1000 for photodecomposition studies (Gonzalez-Fernandez et al. Mol Vis 2016, 22:953), may facilitate studies of metabolically active photoreceptor suspensions or whole retinas in physiological assays.

Ophthalmic genetics in South America.

South America comprises of heterogeneous topographies, populations, and health care systems. Therefore, it is not surprising to see differences among the countries regarding expertise, education, and practices of ophthalmic genetics for patients with rare eye diseases. Nevertheless, common challenges such as limited genetics training in medical schools and among ophthalmologists, scarcity of diagnostic tools for phenotyping, and expensive genetic testing not covered by the public healthcare systems, are seen in all of them. Here, we provide a detailed report of the current status of ophthalmic genetics, described by the personal views of local ophthalmologists from Brazil, Colombia, Argentina, and Chile. By reporting our strengths and weaknesses as a region, we intend to highlight the need for guidelines on how to manage these patients aligned with public health policies. Our region contributes to research worldwide, with thousands of well diagnosed patients from a number of unique and genetically diverse populations. The constant expansion of ophthalmic genetics and molecular diagnostics requires us to join forces to collaborate across South America and with other countries to improve access to next-generation diagnostics and ultimately improve patient care.

Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

Pericellular interphotoreceptor matrix dictates outer retina critical surface tension.

Retinal detachments create two pathological surfaces, the surface of the outer neural retinal, and an apical retinal-pigmented epithelium (RPE) surface. The physicochemical properties of these two new surfaces are poorly understood. At a molecular level little is known how detachments form, how to optimize reattachment, or prevent extension of the detachment. A major limitation is lack of information about the biophysical consequences of the retina-RPE separation. The primary challenge is determining the molecular properties of the pathological interface surfaces. Here, using detached bovine retina, we show that this hurdle can be overcome through a combination of biophysical and ultrastructural approaches. The outer surface of freshly detached bovine neural retina, and isolated molecular components of the outer retina were subjected to: 1) Contact angle goniometry to determine the critical surface tension of the outer retinal surface, isolated insoluble interphotoreceptor matrix (IPM) and purified interphotoreceptor retinoid binding protein (IRBP); 2) Multiple attenuated internal reflectance infrared (MAIR-IR) spectroscopy was used to characterize the molecular composition of the retinal surface. MAIR-IR depth penetration was established through ellipsometric measurement of barium-stearate films. Light microscopy, immunohistochemistry and electron microscopy defined the structures probed spectroscopically. Furthermore, the data were correlated to IR spectra of docosahexaenoic acid, hyaluronan, chondroitin-6-sulfate and IRBP, and imaging by IR-microscopy. We found that the retinal critical surface tension is 24 mN/m, similar to isolated insoluble IPM and lower than IRBP. Barium-stearate calibration studies established that the MAIR-IR spectroscopy penetration depth was 0.2 µm. Ultrastructural observations and MAIR-IR studies of isolated outer retina components determined that the pericellular IPM coating the outer retinal surface is primarily responsible for these surface properties. The critical surface tension of detached bovine retina is dictated not by the outer segments, but by a pericellular IPM covering the outer segment tips.

Technical brief: Pump-probe paradigm in an integrating cavity to study photodecomposition processes.

PURPOSE: Assaying photodecomposition is challenging because light must be used to initiate the photodamage and light must be used to monitor the photodecomposition. The experimental requirements are as follows: 1) During exposure of the actinic beam, continuously monitor the spectral characteristics of the sample, 2) uniformly expose the reactants to the actinic source, 3) obtain informative spectra in the presence of light scatter, and 4) achieve sufficient sensitivity for dilute reactants. Traditional spectrophotometers cannot address these issues due to sample turbidity, the inability to uniformly expose the cuvette contents to the incident beam, the inability to simultaneously perform spectral scans, and inherent low sensitivity. Here, we describe a system that meets these challenges in a practical way. METHODS: Light access to a 8.6 ml quartz integrating sphere containing 10 µM all-trans retinol in PBS was provided by three ports at right angles allowing for the following: 1) actinic light delivery from light-emitting diodes (LEDs) firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec (10,000 µsec scan time) via an OLIS RSM 1000 ultraviolet/visual (UV/Vis) rapid-scanning spectrophotometer (RSM), and 3) light exit to the detector photomultiplier. The RSM spectral intermediate slit was partially covered to allow for a "dark" period of 2,000 µsec when no scanning light was admitted to the cuvette. During that interval, the LED was flashed, and the photomultiplier was temporarily blocked by a perforated spinning shutter disk. The absorbance per centimeter, which is increased due to the internal reflectance of the integrating sphere compared to a standard 1 cm rectangular cuvette, was calculated according to Fry et al. (2010) Applied Optics 49:575. Retinoid photodecomposition was confirmed with high-performance liquid chromatography (HPLC). RESULTS: Using the RSM to trigger the LED flash and photomultiplier shutter closure during the "dark" period allowed actinic flashes to be placed between scans. Exposure of the all-trans retinol to 366 nm flashes resulted in marked reduction in absorbance and a blue shift of the λmax. A white LED, despite its higher photon output, did not support all-trans retinol photolysis. Singular value decomposition (SVD) analysis revealed three spectral intermediates with mechanism, I -> II -> III. HPLC analysis of the reactants at the beginning and the conclusion of the light exposure confirmed the retinol photodecomposition. CONCLUSIONS: The highly reflecting cavity acts as a multipass cuvette that markedly increased the light path length and, thus, sensitivity. Triggering the LED during a dark period within the scan time allowed the actinic flashes to be interleafed between scans in a pump-probe paradigm. Furthermore, the entire sample was exposed to scan beam and actinic flashes, which is not possible in traditional spectrophotometers. Finally, the integrating cavity cuvette allowed use of turbid samples. SVD was useful for resolving spectral intermediates. Although the identity of the intermediates was not determined here, the ability to define molecular intermediates during photodecomposition reactions will allow future studies to isolate and identify the degradation products and determine the mechanism of light-induced retinoid degradation and that of retinoid-binding protein-mediated photoprotection.

Fold conservation and proteolysis in zebrafish IRBP structure: Clues to possible enzymatic function?

Multiple functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) may explain its localization in the retina, vitreous and pineal gland and association with retinitis pigmentosa and myopia. We have been engaged in uncovering the structure-function relationships of this interesting protein long thought to bind visual-cycle retinoids and fatty acids in the subretinal space. Although hydrophobic domains capable of binding such ligands have now been found, we ask what other structural domains might be present that could predict new functions? Interestingly, IRBP possesses a fold similar to C-terminal processing proteases (CTPases) but is missing the PDZ domain. Here we present structural evidence that this fold may have a role in a recently observed autoproteolytic activity of the two-module zebrafish (z) IRBP (Ghosh et al. Exp. Eye Res., 2015). When the structure of Scenedesmus obliquus D1 CTPase (D1P) is superimposed with the first module of zIRBP (z1), the PDZ domain of D1P occupies roughly the same position in the amino acid sequence as the inter-domain tether in z1, between residues P71 and P85. The catalytic triad K397, S372 and E375 of D1P is located at the inter-domain interfacial cleft, similarly as the tetrad K241, S243, D177 and T179 of z1 residues, presumed to have proteolytic function. Packing of two adjacent symmetry-related molecules within the z1 crystal show that the helix α8 penetrates the interfacial cleft underneath the inter-domain tether, forming a simple intermolecular "knot". The full-length zIRBP is cleaved at or immediately after T309, which is located at the end of α8 and is the ninth residue of the second module z2. We propose that the helix α8 within intact zIRBP bends at P301, away from the improbable knotted fold, and positions the cleavage site T309 near the putative catalytic tetrad of the neighboring zIRBP to be proteolytically cleaved. The conservation of this functional catalytic domain suggests that possible physiological roles of IRBP as a hydrolase needs to be considered.

Structure of zebrafish IRBP reveals fatty acid binding.

Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all-trans and 11-cis retinol, and 11-cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP's unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP's. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90 Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket with a single bound molecule of oleic acid was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all-trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding.

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ∼300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP's free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scavenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP's antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space.

Seroprevalence and herd-level associated factors of pathogenic Leptospira spp. circulating locally in dairy cattle in Uruguay.

Leptospirosis is a zoonotic disease of worldwide importance. In Uruguay, it is endemic in cattle and primarily affects people with occupational exposure to livestock. The aim of this study was to determine the national seroprevalence and associated factors of local pathogen Leptospires in dairy cattle. A cross-sectional study was carried out. Herds were stratified by size (1-50, 51-250, and > 250 cattle), and up to 60 dairy cows per herd were randomly selected. A total of 4269 serum samples from 101 dairy herds were analyzed by microscopic agglutination test (MAT). A two-stage sampling design was used to estimate population seroprevalence of Leptospira spp. In order to determine the factors associated with the disease, herds with at least 1 seropositive animal were considered as case herds. Seroprevalence of Leptospira was 27.80% with a 95% CI [21.06, 34.54] at the animal level and 86.92% with a 95% CI [80.00, 93.75] at the herd level. The serology confirms the predominance of serogroups Sejroe and Pomona in our herd with the presence of incidental leptospires infection, in smaller proportion, but with a wide distribution at farm level. The population size and purchasing replacement of cows on dairy farms were associated with infection at farm level. The serologic studies confirmed that exposure to Leptospira spp. is endemic in our herds, and the spreading over dairy herds. Although the movement of purchased females and the size of the herd were associated with the disease, more studies should be conducted, to better understand the epidemiology of the disease and to highlight the possible risks to public health, especially in rural workers, farmers and veterinarians.

Assessment of the Impact of Home-Based Hospitalization on Health Outcomes: An Observational Study.

INTRODUCTION: In Portugal, evidence of clinical outcomes within home-based hospitalization programs remains limited. Despite the adoption of homebased hospitalization services, it is still unclear whether these services represent an effective way to manage patients compared with inpatient hospital care. Therefore, the aim of this study was to evaluate the outcomes of home-based hospitalization compared with conventional hospitalization in a group of patients with a primary diagnosis of infectious, cardiovascular, oncological, or 'other' diseases. METHODS: An observational retrospective study using anonymized administrative data to investigate the outcomes of home-based hospitalization (n = 209) and conventional hospitalization (n = 192) for 401 Portuguese patients admitted to CUF hospitals (Tejo, Cascais, Sintra, Descobertas, and the Unidade de Hospitalização Domiciliária CUF Lisboa). Data on demographics and clinical outcomes, including Barthel index, Braden scale, Morse scale, mortality, and length of hospital stay, were collected. The statistical analysis included comparison tests and logistic regression. RESULTS: The study found no statistically significant differences between patients' admission and discharge for the Barthel index, Braden scale, and Morse scale scores, for both conventional and home-based hospitalizations. In addition, no statistically significant differences were found in the length of stay between conventional and home-based hospitalization, although patients diagnosed with infectious diseases had a longer stay than patients with other conditions. Although the mortality rate was higher in home-based hospitalization compared to conventional hospitalization, the mortality risk index (higher in home-based hospitalization) assessed at admission was a more important predictor of death than the type of hospitalization. CONCLUSION: The study found that there were no significant differences in outcomes between conventional and home-based hospitalization. Home-based hospitalization was found to be a valuable aspect of patient- and family-centered care. However, it is noteworthy that patients with infectious diseases experienced longer hospital stays.

Predicting post-discharge complications in cardiothoracic surgery: A clinical decision support system to optimize remote patient monitoring resources.

Cardiac surgery patients are highly prone to severe complications post-discharge. Close follow-up through remote patient monitoring can help detect adverse outcomes earlier or prevent them, closing the gap between hospital and home care. However, equipment is limited due to economic and human resource constraints. This issue raises the need for efficient risk estimation to provide clinicians with insights into the potential benefit of remote monitoring for each patient. Standard models, such as the EuroSCORE, predict the mortality risk before the surgery. While these are used and validated in real settings, the models lack information collected during or following the surgery, determinant to predict adverse outcomes occurring further in the future. This paper proposes a Clinical Decision Support System based on Machine Learning to estimate the risk of severe complications within 90 days following cardiothoracic surgery discharge, an innovative objective underexplored in the literature. Health records from a cardiothoracic surgery department regarding 5 045 patients (60.8% male) collected throughout ten years were used to train predictive models. Clinicians' insights contributed to improving data preparation and extending traditional pipeline optimization techniques, addressing medical Artificial Intelligence requirements. Two separate test sets were used to evaluate the generalizability, one derived from a patient-grouped 70/30 split and another including all surgeries from the last available year. The achieved Area Under the Receiver Operating Characteristic curve on these test sets was 69.5% and 65.3%, respectively. Also, additional testing was implemented to simulate a real-world use case considering the weekly distribution of remote patient monitoring resources post-discharge. Compared to the random resource allocation, the selection of patients with respect to the outputs of the proposed model was proven beneficial, as it led to a higher number of high-risk patients receiving remote monitoring equipment.

On the use of fractional calculus to improve the pulse arrival time (PAT) detection when using photoplethysmography (PPG) and electrocardiography (ECG) signals.

The pulse arrival time (PAT) has been considered a surrogate measure for pulse wave velocity (PWV), although some studies have noted that this parameter is not accurate enough. Moreover, the inter-beat interval (IBI) time series obtained from successive pulse wave arrivals can be employed as a surrogate measure of the RR time series avoiding the use of electrocardiogram (ECG) signals. Pulse arrival detection is a procedure needed for both PAT and IBI measurements and depends on the proper fiducial points chosen. In this paper, a new set of fiducial points that can be tailored using several optimization criteria is proposed to improve the detection of successive pulse arrivals. This set is based on the location of local maxima and minima in the systolic rise of the pulse wave after fractional differintegration of the signal. Several optimization criteria have been proposed and applied to high-quality recordings of a database with subjects who were breathing at different rates while sitting or standing. When a proper fractional differintegration order is selected by using the RR time series as a reference, the agreement between the obtained IBI and RR is better than that for other state-of-the-art fiducial points. This work tested seven different traditional fiducial points. For the agreement analysis, the median standard deviation of the difference between the IBI and RR time series is 5.72 ms for the proposed fiducial point versus 6.20 ms for the best-performing traditional fiducial point, although it can reach as high as 9.93 ms for another traditional fiducial point. Other optimization criteria aim to reduce the standard deviation of the PAT (7.21 ms using the proposed fiducial point versus 8.22 ms to 15.4 ms for the best- and worst-performing traditional fiducial points) or to minimize the standard deviation of the PAT attributable to breathing (3.44 ms using the proposed fiducial point versus 4.40 ms to 5.12 ms for best- and worst-performing traditional fiducial points). The use of these fiducial points may help to better quantify the beat-to-beat PAT variability and IBI time series.

The First Steps of the Visual Cycle in Human Rod and Cone Photoreceptors.

Purpose: Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Methods: Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360 nm; emission, >420 nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380 nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. Results: We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. Conclusions: The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

Carotenoids as possible interphotoreceptor retinoid-binding protein (IRBP) ligands: a surface plasmon resonance (SPR) based study.

Uptake, transport and stabilization of xanthophylls in the human retina are important components of a complex multistep process that culminates in a non-uniform distribution of these important nutrients in the retina. The process is far from understood; here, we consider the potential role of interphotoreceptor retinoid-binding protein (IRBP) in this process. IRBP is thought to facilitate the exchange of 11-cis-retinal, 11-cis-retinol and all-trans-retinol between the retinal pigment epithelium (RPE), photoreceptors and Müller cells in the visual cycle. Structural and biochemical studies suggest that IRBP has a variety of nonequivalent ligand binding sites that function in this process. IRBP is multifunctional, being able to bind a variety of physiologically significant molecules including fatty acids in the subretinal space. This wide range of binding activities is of particular interest because it is unknown whether the lutein and zeaxanthin found in the macula originate from the choroidal or retinal circulations. If from the choroidal circulation, then IRBP is a likely mediator for their transport across the interphotoreceptor matrix. In this report, we explore the binding interactions of retinoids, fatty acids, and carotenoids with IRBP using surface plasmon resonance (SPR)-based biosensors. IRBP showed similar affinity toward retinoids and carotenoids (1-2 µM), while fatty acids had approximately 10 times less affinity. These results suggest that further studies should be carried out to evaluate whether IRBP has a physiologically relevant role in binding lutein and zeaxanthin in the interphotoreceptor matrix.

Cone outer segment and Müller microvilli pericellular matrices provide binding domains for interphotoreceptor retinoid-binding protein (IRBP).

The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina, but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that bind IRBP, perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE-photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions.

First identification of Strongyloides stercoralis infection in a pet dog in Argentina, using integrated diagnostic approaches.

BACKGROUND: Strongyloides stercoralis is a soil-transmitted intestinal nematode with a complex life cycle that primarily affects humans, non-human primates, dogs, and occasionally cats. This study presents, to the best of our knowledge, the first case of S. stercoralis infection and its genotyping in a domestic dog from Argentina. METHODS: The patient was a female wired-haired Teckel dog exhibiting recurrent coughing. Coproparasitological analysis using the Baermann technique revealed the presence of rhabditiform larvae morphologically compatible with S. stercoralis. To confirm this finding, molecular diagnosis (18S ribosomal RNA) and analysis of the cox1 gene were performed. RESULTS: We identified a haplotype (HP20) that has previously only been related to S. stercoralis infection in dogs, but was found in the present study to be highly related to the haplotype (HP16) of a zoonotic variant and divergent from those previously described from human patients in Argentina. Furthermore, unlike in human cases following treatment with ivermectin, the dog was negative after moxidectin treatment according to polymerase chain reaction of the sampled faeces. CONCLUSIONS: This case report shows the importance of further investigation into potential transmission events and prevalences of S. stercoralis in dogs and humans in South America. The results reported here should also encourage future work that examines different scenarios of infection with S. stercoralis in dogs and humans with the aim of integrating clinical management, diagnosis, treatment and follow-up strategies in the quest for new approaches for the treatment of this disease in animals and humans. The findings support the adoption of a One Health approach, which recognizes the interconnectedness between animal and human health, in addressing parasitic infections such as strongyloidiasis.

Machine Learning Approaches for the Frailty Screening: A Narrative Review.

Frailty characterizes a state of impairments that increases the risk of adverse health outcomes such as physical limitation, lower quality of life, and premature death. Frailty prevention, early screening, and management of potential existing conditions are essential and impact the elderly population positively and on society. Advanced machine learning (ML) processing methods are one of healthcare's fastest developing scientific and technical areas. Although research studies are being conducted in a controlled environment, their translation into the real world (clinical setting, which is often dynamic) is challenging. This paper presents a narrative review of the procedures for the frailty screening applied to the innovative tools, focusing on indicators and ML approaches. It results in six selected studies. Support vector machine was the most often used ML method. These methods apparently can identify several risk factors to predict pre-frail or frailty. Even so, there are some limitations (e.g., quality data), but they have enormous potential to detect frailty early.

Scaling-up digital follow-up care services: collaborative development and implementation of Remote Patient Monitoring pilot initiatives to increase access to follow-up care.

Background: COVID-19 increased the demand for Remote Patient Monitoring (RPM) services as a rapid solution for safe patient follow-up in a lockdown context. Time and resource constraints resulted in unplanned scaled-up RPM pilot initiatives posing risks to the access and quality of care. Scalability and rapid implementation of RPM services require social change and active collaboration between stakeholders. Therefore, a participatory action research (PAR) approach is needed to support the collaborative development of the technological component while simultaneously implementing and evaluating the RPM service through critical action-reflection cycles. Objective: This study aims to demonstrate how PAR can be used to guide the scalability design of RPM pilot initiatives and the implementation of RPM-based follow-up services. Methods: Using a case study strategy, we described the PAR team's (nurses, physicians, developers, and researchers) activities within and across the four phases of the research process (problem definition, planning, action, and reflection). Team meetings were analyzed through content analysis and descriptive statistics. The PAR team selected ex-ante pilot initiatives to reflect upon features feedback and participatory level assessment. Pilot initiatives were investigated using semi-structured interviews transcribed and coded into themes following the principles of grounded theory and pilot meetings minutes and reports through content analysis. The PAR team used the MoSCoW prioritization method to define the set of features and descriptive statistics to reflect on the performance of the PAR approach. Results: The approach involved two action-reflection cycles. From the 15 features identified, the team classified 11 as must-haves in the scaled-up version. The participation was similar among researchers (52.9%), developers (47.5%), and physicians (46.7%), who focused on suggesting and planning actions. Nurses with the lowest participation (5.8%) focused on knowledge sharing and generation. The top three meeting outcomes were: improved research and development system (35.0%), socio-technical-economic constraints characterization (25.2%), and understanding of end-user technology utilization (22.0%). Conclusion: The scalability and implementation of RPM services must consider contextual factors, such as individuals' and organizations' interests and needs. The PAR approach supports simultaneously designing, developing, testing, and evaluating the RPM technological features, in a real-world context, with the participation of healthcare professionals, developers, and researchers.

Novel strategy for subretinal delivery in Xenopus.

PURPOSE: The subretinal space, which borders the retinal pigment epithelium (RPE), photoreceptors, and Müller cells, is an ideal location to deliver genetic vectors, morpholino oligos, and nanopharmaceuticals. Unfortunately, materials injected into the space tend to stay localized, and degenerative changes secondary to retinal detachment limit its usefulness. Furthermore, such injection requires penetration of the sclera, RPE/choroid, or the retina itself. Here, we developed a strategy in Xenopus to utilize the continuity of the brain ventricle and optic vesicle lumen during embryogenesis as a means to access the subretinal space. METHODS: Wild-type and transgenic embryos expressing green fluorescent protein under the rod-opsin promoter were used for optic vesicle and brain ventricle injections. For injection directly into the optic vesicle, embryos were laid on one side in clay troughs. For brain ventricle injections, embryos were placed standing in foxholes cored from agarose dishes. Linear arrays with each embryo positioned dorsal side toward the micromanipulator facilitated high throughput injections. Twenty-five micrometer micropipettes, which were positioned with a micromanipulator or by hand, were used to pressure inject ~1.0 nl of test solution (brilliant blue, India ink, fluorescein isothiocyanate dextran, or 0.04 µm of latex polystyrene microspheres [FluoSpheres®]). FluroSpheres® were particularly useful in confirming successful injections in living embryos. Anesthetized embryos and tadpoles were fixed in 4% paraformaldehyde and cryoprotected for frozen sections, or dehydrated in ethanol and embedded in methacrylate resin compatible with the microspheres. RESULTS: Direct optic vesicle injections resulted in filling of the brain ventricle, contralateral optic vesicle, and central canal. Stages 24 and 25 gave the most consistent results. However, even with experience, the success rate was only ~25%. Targeting the vesicle was even more difficult beyond stage 26 due to the flattening of the lumen. In contrast, brain ventricle injections were easier to perform and had a ~90% success rate. The most consistent results were obtained in targeting the diencephalic ventricle, which is located along the midline, and protrudes anteriorly just under the frontal ectoderm and prosencephalon. An anterior midline approach conveniently accessed the ventricle without disturbing the optic vesicles. Beyond stage 30, optic vesicle filling did not occur, presumably due to closure of the connection between the ventricular system and the optic vesicles. Securing the embryos in an upright position in the agarose foxholes allowed convenient access to the frontal cephalic region. On methacrylate sections, the RPE-neural retina interphase was intact and labeled with the microspheres. As development continued, no distortion or malformation of the orbital structures was detected. In green fluorescent protein (GFP), transgenic embryos allowed to develop to stage 41, retinal FluoSpheres® labeling and photoreceptor GFP expression could be observed through the pupil. On cryosections, it was found that the FluoSpheres® extended from the diencephalon along the embryonic optic nerve to the ventral subretinal area. GFP expression was restricted to rod photoreceptors. The microspheres were restricted to the subretinal region, except focally at the lip of the optic cup, where they were present within the retina; this was presumably due to incomplete formation of the peripheral zonulae adherens. Embryos showed normal anatomic relationships, and formation of eye and lens appeared to take place normally with lamination of the retina into its ganglion cell and the inner and outer nuclear layers. CONCLUSIONS: Diencephalic ventricular injection before stage 31 provides an efficient strategy to introduce molecules into the embryonic Xenopus subretinal space with minimal to the developing eye or retina.