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Marta Para-Prieto,1,2 Raul Martin,1,3,4 Sara Crespo,2 Laura Mena-Garcia,1 Andres Valisena,2 Lisandro Cordero,1 Gloria Gonzalez Fernandez,2 Juan F Arenillas,5 Nieves Tellez,5 Jose Carlos Pastor1,2 1Universidad de Valladolid, Instituto Universitario de Oftalmobiología Aplicada (IOBA Eye Institute), Valladolid, 47011, Spain; 2Hospital Clínico Universitario, Department of Ophthalmology, Valladolid, 47005, Spain; 3Universidad de Valladolid, Departamento de Física Teórica, Atómica y Óptica, Valladolid, 47011, Spain; 4Plymouth University, Faculty of Health and Human Sciences, Plymouth, UK; 5Hospital Clínico Universitario, Department of Neurology, Valladolid, 47005, SpainCorrespondence: Raul MartinIOBA Eye Institute, University of Valladolid, Paseo de Belen, 17, Valladolid, 47011, SpainTel +34 983 184 848Fax +34 983 184 762Email raul@ioba.med.uva.esObjective: To evaluate the agreement between the peripapillary retinal nerve fiber layer (pRNFL) and foveal thickness (FT) measurements among three different spectral domain-optical coherence tomography (SD-OCT) instruments in a sample of multiple sclerosis (MS) patients and a healthy age-matched control group.Methods: An observational cross-sectional study with three groups: healthy subjects and MS patients w/w a previous clinical diagnosis of optic neuritis (ON) was conducted. The pRNFL and FT were measured using three different SD-OCT instruments (OCT PRIMUS 200 and OCT CIRRUS 500 SD-OCT [Carl Zeiss Meditec] and OCT 3D 2000 [Topcon]).Results: Twenty eyes from 10 healthy subjects matched in age with MS patients without a previous history of eye disease and 62 MS eyes from 31 MS patients (29 eyes without history of ON and 33 eyes with history of ON) were enrolled. Healthy subjects and MS patients without ON did not show differences between the pRNFL and FT thickness (P> 0.99) with any of the instruments. However, MS eyes with a previous episode of ON showed thinner pRNFL and FT (P< 0.01). PRIMUS and CIRRUS OCT showed better agreement of the pRNLF and FT in both healthy and MS eyes. However, 3D OCT showed less agreement in the pRNFL measurement with CIRRUS in both healthy and MS eyes.Interpretation: Although OCT is a valuable technology to improve MS patient assessment, differences between devices must be taken into account. It is necessary to create an international group that standardizes the measurement conditions and above all that provides reference bases for normal subjects.Keywords: spectral-domain optical coherence tomography; SD-OCT, multiple sclerosis; MS, retinal nerve fiber layer; RNFL, foveal thickness; FT, variability

Outer membrane vesicles (OMVs) are spherical structures that contain a small fraction of the periplasm of Gram-negative bacteria, surrounded by its outer membrane. They are naturally produced and detached from the bacterial surface, participate in diverse biological processes, and their diameter size is in the range of 10-300 nm. OMVs have gained interest in different applications, such as the development of biosensors, vaccines, protein chips, and the encapsulation of heterologous proteins and peptides expressed by these microorganisms. However, the use of OMVs in these applications is limited due to the low yields and high purification costs. In this study, we produced green fluorescent protein (GFP) encapsulated into OMVs using Escherichia coli JC8031 transformed with pTRC99A-ssTorA-GFP to establish the production and purification route. Results showed that the motility of the strain prevents its immobilization in alginate, which hampers the purification of OMVs. To address this issue, a zeolite-based column was used to chromatographically separate the OMVs from smaller particles. Further experiments will be focused on standardizing the production and purification of OMVs at a scalable level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Torres-Vanegas, Rincon-Tellez, Guzmán-Sastoque, Valderrama-Rincon, Cruz and Reyes.)

Exposure to commonly used anesthetics leads to neurotoxic effects in animal models-ranging from cell death to learning and memory deficits. These neurotoxic effects invoke a variety of molecular pathways, exerting either immediate or long-term effects at the cellular and behavioural levels. However, little is known about the gene expression changes following early neonatal exposure to these anesthetic agents. We report here on the effects of sevoflurane, a commonly used inhalational anesthetic, on learning and memory and identify a key set of genes that may likely be involved in the observed behavioural deficits. Specifically, we demonstrate that sevoflurane exposure in postnatal day 7 (P7) rat pups results in subtle, but distinct, memory deficits in the adult animals that have not been reported previously. Interestingly, when given intraperitoneally, pre-treatment with dexmedetomidine (DEX) could only prevent sevoflurane-induced anxiety in open field testing. To identify genes that may have been altered in the neonatal rats after sevoflurane and DEX exposure, specifically those impacting cellular viability, learning, and memory, we conducted an extensive Nanostring study examining over 770 genes. We found differential changes in the gene expression levels after exposure to both agents. A number of the perturbed genes found in this study have previously been implicated in synaptic transmission, plasticity, neurogenesis, apoptosis, myelination, and learning and memory. Our data thus demonstrate that subtle, albeit long-term, changes observed in an adult animal's learning and memory after neonatal anesthetic exposure may likely involve perturbation of specific gene expression patterns.

Long QT syndrome (LQTS) is an inherited cardiovascular disorder characterized by electrical conduction abnormalities leading to arrhythmia, fainting, seizures, and an increased risk of sudden death. There are over 15 genes involved in causing LQTS, including SNTA1. Here we generated two human-induced pluripotent stem cell (iPSC) lines from two LQT patients carrying a missense mutation in SNTA1 (c.1088A > C). Both lines showed normal morphological properties, expressed pluripotency markers, showed a normal karyotype profile, and had the ability to differentiate into the three germ layers, making them a valuable tool to model LQTS to investigate the pathological mechanisms related to this SNTA1 variant., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Anesthetics have been shown to cause cytotoxicity, cell death, affect neuronal growth and connectivity in animal models; however, their effects on learning and memory remain to be fully defined. Here, we examined the effects of the inhalation anesthetic sevoflurane (SEV)-both in vivo by examining learning and memory in freely behaving animals, and in vitro using cultured neurons to assess its impact on viability, mitochondrial structure, and function. We demonstrate here that neonatal exposure to sub-clinically used concentrations of SEV results in significant, albeit subtle and previously unreported, learning and memory deficits in adult animals. These deficits involve neuronal cell death, as observed in cell culture, and are likely mediated through perturbed mitochondrial structure and function. Parenthetically, both behavioural deficits and cell death were prevented when the animals and cultured neurons were pre-treated with the anesthetic adjuvant Dexmedetomidine (DEX). Taken together, our data provide direct evidence for sevoflurane-induced cytotoxic effects at the neuronal level while perturbing learning and memory at the behavioural level. In addition, our data underscore the importance of adjuvant agents such as DEX that could potentially counter the harmful effects of commonly used anesthetic agents for better clinical outcomes.

Recent animal studies have drawn concerns regarding most commonly used anesthetics and their long-term cytotoxic effects, specifically on the nervous tissue. It is therefore imperative that the search continues for agents that are non-toxic at both the cellular and behavioural level. One such agent appears to be dexmedetomidine (DEX) which has not only been found to be less neurotoxic but has also been shown to protect neurons from cytotoxicity induced by other anesthetic agents. However, DEX's effects on the growth and synaptic connectivity at the individual neuronal level, and the underlying mechanisms have not yet been fully resolved. Here, we tested DEX for its impact on neuronal growth, synapse formation (in vitro) and learning and memory in a rodent model. Rat cortical neurons were exposed to a range of clinically relevant DEX concentrations (0.05-10 µM) and cellular viability, neurite outgrowth, synaptic assembly and mitochondrial morphology were assessed. We discovered that DEX did not affect neuronal viability when used below 10 µM, whereas significant cell death was noted at higher concentrations. Interestingly, in the presence of DEX, neurons exhibited more neurite branching, albeit with no differences in corresponding synaptic puncta formation. When rat pups were injected subcutaneously with DEX 25 µg/kg on postnatal day 7 and again on postnatal day 8, we discovered that this agent did not affect hippocampal-dependent memory in freely behaving animals. Our data demonstrates, for the first time, the non-neurotoxic nature of DEX both in vitro and in vivo in an animal model providing support for its utility as a safer anesthetic agent. Moreover, this study provides the first direct evidence that although DEX is growth permissive, causes mitochondrial fusion and reduces oxygen reactive species production, it does not affect the total number of synaptic connections between the cortical neurons in vitro., (© 2021. The Author(s).)

Anesthetics are deemed necessary for all major surgical procedures. However, they have also been found to exert neurotoxic effects when tested on various experimental models, but the underlying mechanisms remain unknown. Earlier studies have implicated mitochondrial fragmentation as a potential target of anesthetic-induced toxicity, although clinical strategies to protect their structure and function remain sparse. Here, we sought to determine if preserving mitochondrial networks with a non-toxic, short-life synthetic peptide-P110, would protect cortical neurons against both inhalational and intravenous anesthetic-induced neurotoxicity. This study provides the first direct and comparative account of three key anesthetics (desflurane, propofol, and ketamine) when used under identical conditions, and demonstrates their impact on neonatal, rat cortical neuronal viability, neurite outgrowth and synaptic assembly. Furthermore, we discovered that inhibiting Fis1-mediated mitochondrial fission reverses anesthetic-induced aberrations in an agent-specific manner. This study underscores the importance of designing mitigation strategies invoking mitochondria-mediated protection from anesthetic-induced toxicity in both animals and humans.

We used patient dermal fibroblasts to characterize the mitochondrial abnormalities associated with the dilated cardiomyopathy with ataxia syndrome (DCMA) and to study the effect of the mitochondrially-targeted peptide SS-31 as a potential novel therapeutic. DCMA is a rare and understudied autosomal recessive disorder thought to be related to Barth syndrome but caused by mutations in DNAJC19 , a protein of unknown function localized to the mitochondria. The clinical disease is characterized by 3-methylglutaconic aciduria, dilated cardiomyopathy, abnormal neurological development, and other heterogeneous features. Until recently no effective therapies had been identified and affected patients frequently died in early childhood from intractable heart failure. Skin fibroblasts from four pediatric patients with DCMA were used to establish parameters of mitochondrial dysfunction. Mitochondrial structure, reactive oxygen species (ROS) production, cardiolipin composition, and gene expression were evaluated. Immunocytochemistry with semi-automated quantification of mitochondrial structural metrics and transmission electron microscopy demonstrated mitochondria to be highly fragmented in DCMA fibroblasts compared to healthy control cells. Live-cell imaging demonstrated significantly increased ROS production in patient cells. These abnormalities were reversed by treating DCMA fibroblasts with SS-31, a synthetic peptide that localizes to the inner mitochondrial membrane. Levels of cardiolipin were not significantly different between control and DCMA cells and were unaffected by SS-31 treatment. Our results demonstrate the abnormal mitochondria in fibroblasts from patients with DCMA and suggest that SS-31 may represent a potential therapy for this devastating disease., (Copyright © 2019 Machiraju, Wang, Sabouny, Huang, Zhao, Iqbal, King, Prasher, Lodha, Jimenez-Tellez, Ravandi, Argiropoulos, Sinasac, Khan, Shutt and Greenway.)

The genetic cardiomyopathies are a group of disorders related by abnormal myocardial structure and function. Although individually rare, these diseases collectively represent a significant health burden since they usually develop early in life and are a major cause of morbidity and mortality amongst affected children. The heterogeneity and rarity of these disorders requires the use of an appropriate model system in order to characterize the mechanism of disease and develop useful therapeutics since standard drug trials are infeasible. A common approach to study human disease involves the use of animal models, especially rodents, but due to important biological and physiological differences, this model system may not recapitulate human disease. An alternative approach for studying the metabolic cardiomyopathies relies on the use of cellular models which have most frequently been immortalized cell lines or patient-derived fibroblasts. However, the recent introduction of induced pluripotent stem cells (iPSCs), which have the ability to differentiate into any cell type in the body, is of great interest and has the potential to revolutionize the study of rare diseases. In this paper we review the advantages and disadvantages of each model system by comparing their utility for the study of mitochondrial cardiomyopathy with a particular focus on the use of iPSCs in cardiovascular biology for the modeling of rare genetic or metabolic diseases., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

There are adaptive T-cell and antibody autoimmune responses to myelin-derived peptides in multiple sclerosis (MS) and to aquaporin-4 (AQP4) in neuromyelitis optica spectrum disorders (NMOSDs). Strategies aimed at antigen-specific tolerance to these autoantigens are thus indicated for these diseases. One approach involves induction of tolerance with engineered dendritic cells (tolDCs) loaded with specific antigens. We conducted an in-human phase 1b clinical trial testing increasing concentrations of autologous tolDCs loaded with peptides from various myelin proteins and from AQP4. We tested this approach in 12 patients, 8 with MS and 4 with NMOSD. The primary end point was the safety and tolerability, while secondary end points were clinical outcomes (relapses and disability), imaging (MRI and optical coherence tomography), and immunological responses. Therapy with tolDCs was well tolerated, without serious adverse events and with no therapy-related reactions. Patients remained stable clinically in terms of relapse, disability, and in various measurements using imaging. We observed a significant increase in the production of IL-10 levels in PBMCs stimulated with the peptides as well as an increase in the frequency of a regulatory T cell, known as Tr1, by week 12 of follow-up. In this phase 1b trial, we concluded that the i.v. administration of peptide-loaded dendritic cells is safe and feasible. Elicitation of specific IL-10 production by peptide-specific T cells in MS and NMOSD patients indicates that a key element in antigen specific tolerance is activated with this approach. The results warrant further clinical testing in larger trials., Competing Interests: Conflict of interest statement: E.F. and L.S. published an obituary on two leaders in MS [Kildebeck EJ, et al. (2017) The emergence of neuroepidemiology, neurovirology and neuroimmunology: The legacies of John F. Kurtzke and Richard “Dick” T. Johnson. J Neurol 264:817–828]. I.Z. has received travel reimbursement from Genzyme, Biogen, and Merck for national and international meetings over the last 3 y. E.H.M.-L. has received speaker honoraria from Biogen, Roche, Novartis, and Sanofi and a travel reimbursement from Biogen, Roche, Novartis, and Sanofi. E.H.M.-L. has participated in advisory boards for Roche and Sanofi. A.S. has received compensation for consulting services and speaker honoraria from Bayer-Schering, Merck-Serono, Biogen-Idec, Sanofi-Aventis, Teva Pharmaceutical Industries Ltd., Roche, and Novartis. S.L. has received speaker honoraria and travel reimbursement from Biogen, Merck, Novartis, and Teva. I.P.-V. has received travel reimbursement from Roche Spain and Genzyme-Sanofi, European Academy of Neurology, and the European Committee for Treatment and Research in Multiple Sclerosis for international and national meetings over the last 3 y; I.P.-V. holds a patent for an affordable eye-tracking system to measure eye movement in neurologic diseases and holds stock in Aura Innovative Robotics. N.S.-V. received compensation for consulting services and speaker honoraria from Genzyme-Sanofi, Biogen idec, Merck-Serono, and Bayer-Schering. P.V. holds stocks and has received compensation from Bionure Farma SL; Health Engineering SL; Spiral Therapeutics, Inc.; and QMenta SL. L.S. received compensation from Novartis, Celgene, Bionure, Tolerion, Katexco, Atreca, and TG Therapeutics. All other authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: Chronic periodontitis is a multifactorial infectious disease, where multiple bacteria, such as Porphyromonas gingivalis , Prevotella intermedia , and Fusobacterium nucleatum are implicated. The main purpose of researching natural products is to find substances or compounds with antimicrobial activity., Aim: The objective of this work was to determine antimicrobial activity from extracts and obtained fractions from Piper marginatum Jacq and Ilex guayusa Loes on P. gingivalis ATCC 33277, F. nucleatum ATCC 25586, and P. intermedia ATCC 25611., Methods: Total ethanol extracts were obtained from both plants. Fractions were obtained from total ethanol extracts with amberlite as a stationary phase employing hexane, acetone, and ethanol-water as solvents. Qualitative and quantitative phytochemical characterization was performed on total ethanol extracts from both plants. Antimicrobial activity from total ethanol extracts and fractions from both plants were evaluated on P. gingivalis ATCC 33277, F. nucleatum ATCC 25586, and P. intermedia ATCC by the well diffusion method with Wilkins-Chalgren agar., Results: Piper marginatum Jacq total ethanol extract presented antimicrobial activity against all three bacteria, whereas Ilex guayusa Loes was only efficient against P. gingivalis ATCC 33277 and P. intermedia ATCC 25611, with inhibition halos from 9.3 to 30 mm. Ilex guayusa Loes obtained fractions presented antimicrobial activity against all three microorganisms evaluated, with inhibition halos ranging from 9.7 to 18.7 mm. In regards to Piper marginatum Jacq fractions, inhibition halos were between 8.3 and 19 mm, against all three microorganisms evaluated; only hexane fraction did not present antimicrobial activity against F. nucleatum ATCC 25586., Conclusion: Piper marginatum Jacq and Ilex guayusa Loes total ethanol extracts and fractions presented outstanding antimicrobial activity against P. gingivalis ATCC 33277, P. intermedia ATCC 25611, and F. nucleatum ATCC 25586.

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.