Search

Acute liver failure is a rare but serious syndrome, with an incidence of approximately 2,000 to 3,000 cases per year in North America. Its pathophysiology and clinical course vary, depending on the cause of the primary liver injury, and can lead to high morbidity and mortality or the need for liver transplantation, despite available therapies. This syndrome involves excessive activation of the immune system, with damage in other organs, contributing to its high mortality rate. The most accepted definition includes liver injury with hepatic encephalopathy and coagulopathy within the past 26 weeks in a patient with no previous liver disease. The main causes are paracetamol poisoning, viral hepatitis, and drug-induced liver injury, among others. Identifying the cause is crucial, given that it influences prognosis and treatment. Survival has improved with supportive measures, intensive therapy, complication prevention, and the use of medications, such as N-acetylcysteine. Liver transplantation is a curative option for nonresponders to medical treatment, but adequate evaluation of transplantation timing is vital for improving results. Factors such as patient age, underlying cause, and severity of organ failure influence the post-transplant outcomes and survival. Resumen: La falla hepática aguda es un síndrome poco común pero grave, con una incidencia de aproximadamente 2000 a 3000 casos por año en América del Norte. Su fisiopatología y curso clínico varían según la causa del daño hepático primario, y puede llevar a una alta morbimortalidad o necesidad de trasplante hepático, a pesar de las terapias disponibles. Este síndrome involucra una activación excesiva del sistema inmunológico con daño en otros órganos, lo que contribuye a su alta tasa de mortalidad. La definición más aceptada incluye daño hepático con EH y coagulopatía en las últimas 26 semanas en un paciente sin enfermedad hepática previa. Las principales causas son intoxicación por paracetamol, hepatitis viral, lesión hepática inducida por drogas, entre otras. Es crucial identificar la causa, ya que influye en el pronóstico y tratamiento. La supervivencia ha mejorado con medidas de soporte, terapia intensiva, prevención de complicaciones y el uso de medicamentos como la N-acetilcisteína. El trasplante hepático es una opción curativa para casos no respondedores al tratamiento médico, pero la evaluación adecuada del momento para el trasplante es crucial para mejorar los resultados. Factores como la edad del paciente, la causa subyacente y la gravedad de las fallas orgánicas influyen en los resultados y la supervivencia post-trasplante.

Resumen: La falla hepática aguda es un síndrome poco común pero grave, con una incidencia de aproximadamente 2,000 a 3,000 casos por año en América del Norte. Su fisiopatología y curso clínico varían según la causa del daño hepático primario, y puede llevar a una alta morbimortalidad o necesidad de trasplante hepático, a pesar de las terapias disponibles. Este síndrome involucra una activación excesiva del sistema inmunológico con daño en otros órganos, lo que contribuye a su alta tasa de mortalidad. La definición más aceptada incluye daño hepático con encefalopatía hepática y coagulopatía en las últimas 26 semanas en un paciente sin enfermedad hepática previa. Las principales causas son intoxicación por paracetamol, hepatitis viral, lesión hepática inducida por drogas, entre otras. Es crucial identificar la causa, ya que influye en el pronóstico y tratamiento. La supervivencia ha mejorado con medidas de soporte, terapia intensiva, prevención de complicaciones y el uso de medicamentos como la N-acetilcisteína. El trasplante hepático es una opción curativa para casos no respondedores al tratamiento médico, pero la evaluación adecuada del momento para el trasplante es crucial para mejorar los resultados. Factores como la edad del paciente, la causa subyacente y la gravedad de las fallas orgánicas influyen en los resultados y la supervivencia postrasplante. Abstract: Acute liver failure is a rare but serious syndrome, with an incidence of approximately 2,000 to 3,000 cases per year in North America. Its pathophysiology and clinical course vary, depending on the cause of the primary liver injury, and can lead to high morbidity and mortality or the need for liver transplantation, despite available therapies. This syndrome involves excessive activation of the immune system, with damage in other organs, contributing to its high mortality rate. The most accepted definition includes liver injury with hepatic encephalopathy and coagulopathy within the past 26 weeks in a patient with no previous liver disease. The main causes are paracetamol poisoning, viral hepatitis, and drug-induced liver injury, among others. Identifying the cause is crucial, given that it influences prognosis and treatment. Survival has improved with supportive measures, intensive therapy, complication prevention, and the use of medications, such as N-acetylcysteine. Liver transplantation is a curative option for nonresponders to medical treatment, but adequate evaluation of transplantation timing is vital for improving results. Factors such as patient age, underlying cause, and severity of organ failure influence the post-transplant outcomes and survival.

Hepatitis C virus (HCV) infection is a worldwide public health problem associated with significant morbidity and mortality. In the context of liver transplantation, the demand for organs continues to exceed the supply, prompting the consideration of using organs from HCV-positive donors in HCV-negative recipients. The introduction of direct-acting antivirals (DAAs), which have demonstrated great efficacy in eradicating the virus, has made transplantation of organs from donors with HCV infection possible. The present article provides a brief review of the current evidence on the use of organs from HCV-infected patients. Resumen: La infección por el virus de hepatitis C (VHC) es un problema de salud pública a nivel mundial asociado a una gran morbilidad y mortalidad. En el caso del trasplante hepático, la demanda de órganos continúa siendo mayor que la oferta. Este problema ha llevado a tomar en consideración el uso de órganos de donadores con VHC positivos en receptores negativos.Gracias al advenimiento de los antivirales de acción directa (AAD), los cuales han mostrado ser altamente efectivos para la curación del virus, ha sido posible procurar y utilizar órganos de donadores con infección por VHC para ser trasplantados. En este artículo se hace una breve revisión de la evidencia actual del uso de órganos provenientes de pacientes infectados por VHC.

Resumen: La infección por el virus de la hepatitis C (VHC) es un problema de salud pública a nivel mundial asociado a una gran morbimortalidad. En el caso del trasplante hepático, la demanda de órganos continúa siendo mayor que la oferta. Este problema ha llevado a tomar en consideración el uso de órganos de donadores con VHC positivos en receptores negativos.Gracias al advenimiento de los antivirales de acción directa (AAD), los cuales han mostrado ser altamente efectivos para la curación del virus, ha sido posible procurar y utilizar órganos de donadores con infección por el VHC para ser trasplantados. En este artículo se hace una breve revisión de la evidencia actual del uso de órganos provenientes de pacientes infectados por el VHC. Abstract: Hepatitis C virus (HCV) infection is a worldwide public health problem associated with significant morbidity and mortality. In the context of liver transplantation, the demand for organs continues to exceed the supply, prompting the consideration of using organs from HCV-positive donors in HCV-negative recipients. The introduction of direct-acting antivirals (DAAs), which have demonstrated great efficacy in eradicating the virus, has made transplantation of organs from donors with HCV infection possible. The present article provides a brief review of the current evidence on the use of organs from HCV-infected patients.

Los tumores neuroendocrinos pancreáticos representan del 2-10 % de todos los tumores del páncreas y aproximadamente el 7 % de todos los tumores neuroendocrinos. Estos se clasifican como funcionales o no funcionales según la presencia o ausencia de síndromes clínicos asociados con la hipersecreción hormonal. Los insulinomas son los tumores neuroendocrinos pancreáticos funcionales más frecuentes (45 % de los casos) y la causa más frecuente de hipoglucemia hiperinsulinémica endógena persistente en adultos. Además, el 10 % de los tumores neuroendocrinos pancreáticos se asocian con neoplasia endocrina múltiple tipo 1. La insulinomatosis es una entidad clínica distinta en la que existen múltiples insulinomas. Objetivos: exponer los casos reportados hasta el momento de insulinomatosis y describir las causas genéticas, las características clínicas, el tratamiento, y el pronóstico de la insulinomatosis. Métodos: se realizó una búsqueda sobre insulinomatosis y los factores que controlan la proliferación de las células ? en las bases de datos PubMed, Medline y Google Scholar hasta Julio 2020. Resultados: 108 casos con insulinomatosis se han reportado hasta la fecha, siendo recurrente y rara vez malignos. Múltiples protooncogenes y supresores de tumores controlan de forma local y sistémica el crecimiento de las células ?; sin embargo, solo la mutación de MafA en p.Ser64Phe ha sido asociada. Conclusión: la insulinomatosis se caracteriza por la aparición sincrónica y metacrónica de insulinomas. Tiene un fenotipo histológico, clínico y genético diferente a los tumores neuroendocrinos pancreáticos; la mutación MEN-1 es negativa; puede ser esporádica o hereditaria; y MafA podría ser una mutación conductora.

La enfermedad inflamatoria intestinal es una patología crónica, con una incidencia cada vez mayor. Dentro de este grupo de afecciones, la enfermedad de Crohn y la colitis ulcerosa son las más frecuentes. La enterografía por tomografía computada (ETC) y por resonancia magnética (ERM) son las modalidades de elección para la evaluación y seguimiento de la entidad, permitiendo examinar la apariencia de la mucosa, la pared intestinal, las manifestaciones extraintestinales y las complicaciones asociadas. La elección del estudio debe hacerse de acuerdo con la condición clínica de cada paciente.

The effect of initial plastic strain cycle fatigue lives of HSLA specimens is investigated. Specimens were monotonically, quasi-statically loaded under strain control in tension to produce plastic strains (prestrain conditions s0 = 0, s1 = 4,6, s2 = 35,6 and s3 = 66,7 % deformation). Surface analysis on the annealed Zn coat on the prestrain surfaces shows that, the higher the deformation the bigger and deeper the cracks on the Zn coat. Results show that the higher prestrain the higher fatigue resistance for all stress applied, specimens with s3 shows the highest fatigue resistance for these experimental conditions.

Abstract Prolactin (PRL) hormone functions as a pleiotropic cytokine with a protective role in the retina. We recently identified by transcriptome profiling that PRL is one of the most highly upregulated mRNAs in the retinas of mutant rcd1 (PDE6B) and xlpra2 (RPGR) dogs at advanced stages of photoreceptor disease. In the present study, we have identified the expression of a short PRL isoform that lacks exon 1 in canine retinas and analyzed the time-course of expression and localization of this isoform in the retinas of these two models. Using laser capture microdissection to isolate RNA from each of the retinal cellular layers, we found by qPCR that this short PRL isoform is expressed in photoreceptors of degenerating retinas. We confirmed by in situ hybridization that its expression is localized to the outer nuclear layer and begins shortly after the onset of disease at the time of peak photoreceptor cell death in both models. PRL protein was also detected only in mutant dog retinas. Our results call for further investigations into the role of this novel PRL isoform in retinal degeneration.

Purpose: To present a methodology for quantification of the canine retinal vasculature imaged by optical coherence tomography angiography (OCTA) and validate this approach by comparison with fluorescein angiography (FA) and confocal imaging of retinal wholemounts labelled by immunohistochemistry (IHC).Methods: Six normal adult dogs underwent retinal OCTA imaging in both eyes. The images extracted from the different microvascular plexuses at eight retinal locations spanning the central and mid-peripheral fundus were analyzed using the AngioTool software. FA was performed in one eye and was compared to the OCTA images. Six eyes from three dogs were processed by IHC to examine the retinal vasculature.Results: A total of four retinal plexuses were identified by OCTA in the canine retina, and their density and topographical pattern varied with eccentricity. OCTA offered improved resolution over FA with the advantage of allowing imaging of the individual plexuses. Detection by OCTA of small vessels within the deep capillary plexus was possible and approached the level of resolution achieved with ex vivo imaging of the retinal vasculature by confocal microscopy/IHC. The plexuses herein described are analogous to human retinal vasculature.Conclusion: OCTA can be used to image and quantify non-invasively the vascular retinal networks of the canine retina. We provide normative data in eight different retinal locations that can be imaged non-invasively with this technology. This could support analysis of retinal vascular changes associated with disease and following therapeutic intervention.

In vivo imaging of inflammation is crucial for detection and monitoring of many pathologies and noninvasive macrophage quantification has been suggested as a possible approach. Here Keliher et al. describe novel polyglucose nanoparticle tracers that are rapidly excreted by the kidney and with high affinity for macrophages in atherosclerotic plaques.

Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA.

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had "negative-type" mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.

The transition from healthy myocardium to hypertensive heart disease is characterized by a series of poorly understood changes in myocardial tissue microstructure. Incremental alterations in the orientation and integrity of myocardial fibers can be assessed using advanced ultrasonic image analysis. We used a modified algorithm to investigate left ventricular myocardial microstructure based on analysis of the reflection intensity at the myocardial-pericardial interface on B-mode echocardiographic images. We evaluated the extent to which the novel algorithm can differentiate between normal myocardium and hypertensive heart disease in humans as well as in a mouse model of afterload resistance. The algorithm significantly differentiated between individuals with uncomplicated essential hypertension (N = 30) and healthy controls (N = 28), even after adjusting for age and sex (P = 0.025). There was a trend in higher relative wall thickness in hypertensive individuals compared to controls (P = 0.08), but no difference between groups in left ventricular mass (P = 0.98) or total wall thickness (P = 0.37). In mice, algorithm measurements (P = 0.026) compared with left ventricular mass (P = 0.053) more clearly differentiated between animal groups that underwent fixed aortic banding, temporary aortic banding, or sham procedure, on echocardiography at 7 weeks after surgery. Based on sonographic signal intensity analysis, a novel imaging algorithm provides an accessible, non-invasive measure that appears to differentiate normal left ventricular microstructure from myocardium exposed to chronic afterload stress. The algorithm may represent a particularly sensitive measure of the myocardial changes that occur early in the course of disease progression.

Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA), RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC) of antibiotics much faster than the conventional optical density (OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research.

Mutations in the BEST1 gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for BEST1-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (cmr), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when BEST1 and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or BEST1 transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies.

We used quantitative real-time PCR to examine the expression of 112 genes related to retinal function and/or belonging to known pro-apoptotic, cell survival, and autophagy pathways during photoreceptor degeneration in three early-onset canine models of human photoreceptor degeneration, rod cone dysplasia 1 (rcd1), X-linked progressive retinal atrophy 2 (xlpra2), and early retinal degeneration (erd), caused respectively, by mutations in PDE6B, RPGRORF15, and STK38L. Notably, we found that expression and timing of differentially expressed (DE) genes correlated with the cell death kinetics. Gene expression profiles of rcd1 and xlpra2 were similar; however rcd1 was more severe as demonstrated by the results of the TUNEL and ONL thickness analyses, a greater number of genes that were DE, and the identification of altered expression that occurred at earlier time points. Both diseases differed from erd, where a smaller number of genes were DE. Our studies did not highlight the potential involvement of mitochondrial or autophagy pathways, but all three diseases were accompanied by the down-regulation of photoreceptor genes, and up-regulation of several genes that belong to the TNF superfamily, the extrinsic apoptotic pathway, and pro-survival pathways. These proteins were expressed by different retinal cells, including horizontal, amacrine, ON bipolar, and Müller cells, and suggest an interplay between the dying photoreceptors and inner retinal cells. Western blot and immunohistochemistry results supported the transcriptional regulation for selected proteins. This study highlights a potential role for signaling through the extrinsic apoptotic pathway in early cell death events and suggests that retinal cells other than photoreceptors might play a primary or bystander role in the degenerative process.

There is a long history and a growing interest in the canine as a subject of study in neuroscience research and in translational neurology. In the last few years, anatomical and functional magnetic resonance imaging (MRI) studies of awake and anesthetized dogs have been reported. Such efforts can be enhanced by a population atlas of canine brain anatomy to implement group analyses. Here we present a canine brain atlas derived as the diffeomorphic average of a population of fifteen mesaticephalic dogs. The atlas includes: 1) A brain template derived from in-vivo, T1-weighted imaging at 1 mm isotropic resolution at 3 Tesla (with and without the soft tissues of the head); 2) A co-registered, high-resolution (0.33 mm isotropic) template created from imaging of ex-vivo brains at 7 Tesla; 3) A surface representation of the gray matter/white matter boundary of the high-resolution atlas (including labeling of gyral and sulcal features). The properties of the atlas are considered in relation to historical nomenclature and the evolutionary taxonomy of the Canini tribe. The atlas is available for download (https://cfn.upenn.edu/aguirre/wiki/public:data_plosone_2012_datta).

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.

El Laboratorio Genes Ltda, Laboratorio Nacional de Referencia para los laboratorios que realizan pruebas de paternidad/maternidad con marcadores de ADN, ha diseñado y administrado el Ejercicio Control de Calidad 2009, invitando a participar laboratorios nacionales y extranjeros acreditados y no acreditados con la Norma ISO 17025. El ejercicio constó de una parte práctica y una teórica, para la práctica se remitió 3 muestras de referencia no relacionadas biológicamente entre sí, en forma de manchas de sangre en Tarjetas FTA para ser tipificada por cada laboratorio participante con los métodos y marcadores que habitualmente utiliza en su rutina; para la parte teórica se enviaron tres casos con los perfiles genéticos para que los participantes calcularan los parámetros forenses indicados en el instructivo que se adjunto, para estos cálculos se adjuntó las bases de datos de referencia para los marcadores indicados y que utiliza el Laboratorio de Referencia, de estos tres ejercicios teóricos uno fue obligatorio y los otros dos opcionales. Un total de 23 laboratorios se inscribieron, de ellos 22 participaron en el ejercicio enviando resultados, uno de República Dominicana, dos de Panamá, dos de Perú, dos de Ecuador, uno de Brasil y 14 de Colombia, convirtiendo este ejercicio en una actividad internacional reconocida.

RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean +/- standard deviation [SD] = 0.07% +/- 0.06% and volume = 1.3 +/- 0.6 cm(3)). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% +/- 0.06%) and volume (8.2 +/- 0.8 cm(3)) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1-4 y of age. Human RPE65-LCA patients (ages 18-23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 +/- 0.5 mm) was within the normal range (3.2 +/- 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 +/- 1.2 cm(3)) compared to controls (29.7 +/- 8.3 cm(3), p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 +/- 11.1 cm(3)) was comparable to normal (48.8 +/- 3.1 cm(3), p = 0.2) with higher intensity light stimulation.Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.

The use of a new subretinal injection device (RetinaJect™ Subretinal Cannula, SurModics, Inc., Eden Prairie, MN) to access the subretinal space in the canine model was evaluated. Subretinal injections were performed in 33 mongrel dogs between 2 and 52 months of age (median = 9 months). In 5 normal dogs the injection of 150 μl saline or India ink occurred by using a conventional subretinal injection device (CSID) with a 30-gauge anterior chamber irrigating cannula. The sclera had to be surgically exposed and penetrated before the subretinal injection with the CSID could occur. After removing the CSID, the conjunctiva over the sclerotomy site had to be closed. In a second group of 28 dogs [16 normals, 10 RPE65 mutants, and 2 with progressive rod cone degeneration (prcd)], the 25-gauge needle of the RetinaJect™ was used to penetrate the conjunctiva and the sclera. Once the tip of the needle was close to the retinal surface, a 39-gauge polyimide cannula was extended and brought into apposition with the retina for the subsequent subretinal injection of 150 μl saline, India ink, or adeno-associated virus (AAV). No closure of the conjunctiva was required. The animals were clinically monitored between 1 and 59 weeks after surgery. From this second group 25 eyes were harvested for routine histological analysis either immediately after surgery or after a clinical observation time of between 1 and 40 weeks. Both devices provided equally successful access to the subretinal space. The main advantage of the RetinaJect™ was that no surgical dissection was required; this led to a shorter procedure time and milder postoperative conjunctival swelling. In summary, the use of the RetinaJect™ can be recommended as an alternative to the CSID for subretinal injections in dogs.

Abstract PRLΔE1, a retina-specific isoform of prolactin, is expressed in multiple and diverse forms of canine inherited retinal degeneration (IRD). We find that while PRLΔE1 expression in rods is not associated with the initial phase of disease characterized by acute photoreceptor cell death, it is associated with the protracted phase of slow cell loss. Restoration of photoreceptors to a healthy state by gene-specific replacement therapy of individual IRDs successfully suppresses PRLΔE1 expression. Moreover, short-term PRLΔE1 silencing using shRNA results in preservation of outer nuclear layer thickness, suggesting PRLΔE1 drives retinal disease. However, longer-term observations reveal off-target toxic effects of the PRLΔE1 shRNA, precluding determination of its full therapeutic potential. Future research efforts aimed at enhancing the safety and specificity of PRLΔE1-targeting strategies may identify a potential universal intervention strategy for sustaining photoreceptors during the prolonged phase of multiple IRDs.

Mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRorf15) cause X-linked retinitis pigmentosa, a severe and early onset inherited retinal degeneration. The underlying pathogenic mechanisms and variability in disease severity remain to be fully elucidated. The present study examines structural features of the ORF15 exonic region to provide new insights into the disease pathogenesis. Using canine and human RNA samples, we identified several novel RPGR ORF15-like linear RNA transcripts containing cryptic introns (exitrons) within the annotated exon ORF15. Furthermore, using outward-facing primers designed inside exitrons in the ORF15 exonic region, we found many of previously unidentified circular RNAs (circRNAs) that formed via back fusion of linear parts of the RPGRorf15 pre-mRNAs. These circRNAs (resistant to RNAse R treatment) were found in all studied cells and tissues. Notably, some circRNAs were present in cytoplasmic and polysomal RNA fractions. Although certain RPGR circRNAs may be cell type specific, we found some of the same circRNAs expressed in different cell types, suggesting similarities in their biogenesis and functions. Sequence analysis of RPGR circRNAs revealed several remarkable features, including identification of N6-methyladenosine (m6A) consensus sequence motifs and high prevalence of predictive microRNA binding sites pointing to the functional roles of these circRNAs. Our findings also illustrate the presence of non-canonical RPGR circRNA biogenesis pathways independent of the known back splicing mechanism. The obtained data on novel RPGR circRNAs further underline structural complexity of the RPGR ORF15 region and provide a potential molecular basis for the disease phenotypic heterogeneity.

Importance: Some medications have effects that depend on the time of day they are given. Current knowledge of the time-of-day effects of specific medications in hospitalized patients with cardiovascular disease is very limited. In hospitalized patients, increased medication efficiency might reduce dose (and associated side effects) and/or the length of time in the Intensive Care Unit (ICU) or hospital–potentially improving patient outcomes and patient and family quality of life and reducing financial costs. We studied whether the time of day or night patients in Cardiac or Intensive Care Units receive a diuretic affects urine volume.Methods: In this observational study, data were collected from 7,685 patients (63% male, 18 to 98 years old) admitted to one hospital’s Acute Care Cardiac units, Cardiac ICUs, Cardiac Surgery ICUs, and/or Non-cardiac ICUs who received intravenous furosemide (a diuretic), had measurements of urine volume, were hospitalized for ≥3 days between January 2016 to July 2021 and were older than 18 years. The outcomes of interest were urine volume normalized by the most recent (not older than 24 h) weight or body mass index (BMI), (i) in the hour after the time of diuretic administration, and (ii) when no diuretics were administered for the previous 3 h.Results: We identified diuretic medication administration time 23:00–04:59 as a predictor of higher urine volume response. For patients without recent diuretic medication, higher urine volume was predicted 11:00–16:59 and 17:00–22:59. Other factors that affected urine volume response to the diuretic were sex, age, medication dose, creatinine concentration, diagnoses, and hospital unit.Discussion: Time-of-day of medication administration may be a factor associated with increased medication efficiency. Randomized controlled trials should be conducted to quantify the relative effect of modifiable factors, such as time of medication administration, that may affect short- and longer-term outcomes.

Photoreceptors possess a highly specialized primary cilium containing expanded ciliary membrane discs called the outer segment. The photoreceptor cilium is essential for the maintenance of the outer segment, and pathogenic variants in more than 50 cilia-related genes have been identified as causing non-syndromic inherited retinal diseases in patients. The retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) is a structural protein localized to the photoreceptor cilium and biallelic RPGRIP1 variants have been associated with non-syndromic human inherited retinal diseases. In a canine cone-rod dystrophy model, a naturally occurring 44-bp exonic insertion in RPGRIP1 (RPGRIP1ins44/ins44) is the primary disease locus while an additional homozygous variant in MAP9 (microtubule associated protein 9) (MAP9aff/aff) acts as a modifier associated with early disease onset. MAP9 was originally identified as a microtubule-binding protein stabilizing microtubule structure during both mitosis and interphase in human cell lines. However, the roles of MAP9 in primary cilia, including photoreceptor neurosensory cilia, have not been well understood. Hence, we characterized the pathogenic phenotypes associated with homozygous MAP9 variant, and investigated the molecular function of MAP9 in primary cilia using the RPGRIP1-associated oligogenic canine cone-rod dystrophy model as well as cultured cells. Both functionally and structurally, the RPGRIP1ins44/ins44MAP9aff/aff retina exhibited progressive cone photoreceptor degeneration starting earlier than the retina affected by RPGRIP1ins44/ins44 alone. Based on immunostaining of canine retinal sections and cultured cells, we found that MAP9 is prominently localized in the basal body of primary cilia and played an important role in maintaining the structure of ciliary microtubule axoneme. These findings suggest that the affected MAP9, together with mutant RPGRIP1, is deprived of critical roles in cilia organization and maintenance resulting in altered cilia structure and function giving rise to early onset and accelerated disease progression in the RPGRIP1ins44/ins44MAP9aff/aff double homozygote cone-rod dystrophy canine model.

Inflammation is a protective response of the body. Here, authors show that healthy inflammation induces remarkably consistent changes in white cell and platelet populations, regardless of the underlying cause, including heart attack, infection and trauma.

Background and Objectives: During the COVID-19, the demand for non-invasive ventilatory support equipment significantly increased. In response, a novel non-invasive ventilatory support model called CPAP-AirFlife™ was developed utilizing existing technologies. This model offers technological advantages, including an aerosol-controlled helmet suitable for high-risk environments such as ambulances. Additionally, it is cost-effective and does not require medical air, making it accessible for implementation in low-level hospitals, particularly in rural areas. This study aimed to assess the efficacy of CPAP-AirFlife™ by conducting a non-inferiority comparison with conventional ventilation equipment used in the Intensive Care Unit. Materials and Methods: A clinical study was conducted on normal subjects in a randomized and sequential manner. Parameters such as hemoglobin oxygen saturation by pulse oximetry, exhaled PCO2 levels, vital signs, and individual tolerance were compared between the CPAP-AirFlife™ and conventional equipment. The study population was described in terms of demographic characteristics and included in the analysis. Results: It was shown that the CPAP-AirFlife™ was not inferior to conventional equipment in terms of efficacy or tolerability. Hemoglobin oxygen saturation levels, exhaled PCO2 levels, vital signs, and individual tolerance did not significantly differ between the two models. Conclusions: The findings suggest that CPAP-AirFlife™ is a practical and cost-effective alternative for non-invasive ventilatory support. Its technological advantages, including the aerosol-controlled helmet, make it suitable for high-risk environments. The device’s accessibility and affordability make it a promising solution for implementation in low-level hospitals, particularly in rural areas. This study supports using CPAP-AirFlife™ as a practical option for non-invasive ventilatory support, providing a valuable contribution to respiratory care during the COVID-19 pandemic and beyond.

Supervised machine learning applications in health care are often limited due to a scarcity of labeled training data. To mitigate the effect of small sample size, we introduce a pre-training approach, Patient Contrastive Learning of Representations (PCLR), which creates latent representations of electrocardiograms (ECGs) from a large number of unlabeled examples using contrastive learning. The resulting representations are expressive, performant, and practical across a wide spectrum of clinical tasks. We develop PCLR using a large health care system with over 3.2 million 12-lead ECGs and demonstrate that training linear models on PCLR representations achieves a 51% performance increase, on average, over six training set sizes and four tasks (sex classification, age regression, and the detection of left ventricular hypertrophy and atrial fibrillation), relative to training neural network models from scratch. We also compared PCLR to three other ECG pre-training approaches (supervised pre-training, unsupervised pre-training with an autoencoder, and pre-training using a contrastive multi ECG-segment approach), and show significant performance benefits in three out of four tasks. We found an average performance benefit of 47% over the other models and an average of a 9% performance benefit compared to best model for each task. We release PCLR to enable others to extract ECG representations at https://github.com/broadinstitute/ml4h/tree/master/model_zoo/PCLR.

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 μL, 3–10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal. Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified – with cell-type resolution – the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection. Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors. Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research.

Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance.We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ∼3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ∼6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1dci). PPT1dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n = 4) and homozygous wild-type (n = 1) in cases either homozygous for PPT1dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1dci, and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1dci variant. Therefore, we propose PPT1dci as causative for a non-syndromic form of PRA (PRAPPT1) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.

The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi (S. Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2, and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2, the amount of the OmpC porin decreased substantially, whereas in individual cse2, cas6e, cas1, or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S. Typhi cse2, cas5, cas6e, cas1, cas2, and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S. Typhi.

Background Neutrophils are thought to be short‐lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single‐cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady‐state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late‐emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecFHI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post‐MI. Validation studies using quantitative polymerase chain reaction of fluorescent‐activated cell sorter sorted Ly6G+SiglecFHI and Ly6G+SiglecFLO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA‐deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G+SiglecFHI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G+SiglecFHI neutrophils expressed high levels of Myc‐regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G+SigF+(Myc+NFϰB+) neutrophil that has been overlooked because of negative selection.

BackgroundPrevious studies have demonstrated that statin therapy improves cardiac outcomes, probably by stabilizing thin‐cap fibroatheroma in patients with coronary artery disease. However, major adverse cardiac events still occur in some patients, despite statin therapy. The aim of this study is to identify clinical predictors for the lack of a favorable vascular response to statin therapy in patients with coronary artery disease. Methods and ResultsA total of 140 nonculprit plaques from 84 patients with coronary artery disease who were treated with a statin and had serial optical coherence tomography imaging (median interval, 6.3 months) were included. Thin‐cap area (fibrous cap thickness,

We have analyzed the complex pattern of the inflammatory response in early-onset canine models of human retinitis pigmentosa, rcd1, xlpra2 and erd, as well as late-onset xlpra1, in comparative manner. The time course of immune response genes and proteins expression was examined along the timeline of photoreceptors degeneration. Gene expression analysis of the early-onset models prior to and after the peak of photoreceptors death identified the involvement of multiple immune response genes including those encoding constituents of the NLRP3 inflammasome, its substrates, pro-IL1B, pro-IL18, and common components of IL1B, IL18 and TLR4 pathways. Out of two activated caspase-1 cleavage products, IL1B and IL18, only IL1B was detected in rcd1 and xlpra2 while precursor IL18 remained unprocessed in the same protein extract highlighting prominence of IL1B pathway. An overall immune response was most prominent in rcd1 followed by xlpra2 and least prominent in erd. Noticeably, in rcd1 and xlpra2, but not in erd, early induction of the immune response was accompanied by sustained intraretinal migration and activation of retinal microglia. Lastly, delayed activation of the anti-inflammatory factors in all early-onset models was insufficient to counterbalance rapidly progressing inflammation. In contrast to early-onset models, in late-onset xlpra1 retinas a subset of the pro-inflammatory genes was highly upregulated long before any disease-related structural changes occurred, but was counterbalanced by an adequate anti-inflammatory response. Results point out to upregulated immune response accompanying disease progression in animal models of retinal degeneration, and to potential benefits of early anti-inflammatory therapy.

PURPOSE:To examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHO dog). METHODS:The left eyes of T4R RHO dogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure. RESULTS:A brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3. CONCLUSION:The UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration.

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations.