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The choroid, located between the retina and the sclera, is a vascularized and pigmented connective tissue, playing a crucial role in providing oxygen and nutrients to the outer layers of the retina, and in absorbing excessive light. How choroidal melanocytes (CMs) participate in tissue homeostasis through paracrine signaling with neighboring cells is poorly understood. In this study, using two-dimensional and three-dimensional models, we aimed to identify proteins secreted by CMs under different oxidative stress conditions. To do so, CMs, choroidal fibroblasts (CFs), and retinal pigment epithelial (RPE) cells were isolated from native human RPE/choroidal tissues and expanded. RNA was isolated and processed for gene profiling analysis. The self-assembly approach of tissue engineering was used to form 3D stromal substitutes, and RPE cells and/or CMs were added to produce 3D models with different cell combinations. The medium conditioned by cells in 2D and 3D cultures was collected in a non-stressed condition and following acute or chronic oxidative stress exposures, then proteome and ELISA analyses were performed to identify cytokines secreted majorly by CMs. RNA analysis revealed 15 secretome-related transcripts that were more abundantly expressed in CMs compared to the other 2 cell types, including serpin family F member 1 (SERPINF1) (coding for pigment epithelium-derived factor; PEDF) and secreted phosphoprotein 1 (SPP1) (coding for osteopontin). At the protein level, the expression of osteopontin and PEDF was higher in CMs of different age groups compared to CFs and RPE cells. In the 3D models containing CMs, cytokine arrays also identified macrophage inflammatory protein (MIP)-1α/MIP-1β in non-stressed, MIP-1α/MIP-1β, interleukin (IL)-24, and angiogenin following an acute oxidative stress, and macrophage migration inhibitory factor (MIF), granulocyte-colony stimulating factor (G-CSF), intercellular adhesion molecule-1 (ICAM-1), and IL-1α following a chronic oxidative stress. This study identifies for the first time trophic factors secreted by CMs that could influence neighboring cells through paracrine signaling. Of those, PEDF and osteopontin are antioxidative proteins that are known to attenuate oxidative stress damage. Identifying factors that can help manage oxidative stress in the posterior segment of the eye may lead to promising treatments for retinal diseases., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The corneal endothelium is responsible for pumping fluid out of the stroma in order to maintain corneal transparency, which depends in part on the expression and activity of sodium-potassium pumps. In this study, we evaluated how physiologic pressure and flow influence transcription, protein expression, and activity of Na+/K+-ATPase. Native and engineered corneal endothelia were cultured in a bioreactor in the presence of pressure and flow (hydrodynamic culture condition) or in a Petri dish (static culture condition). Transcription of ATP1A1 was assessed using qPCR, the expression of the α1 subunit of Na+/K+-ATPase was measured using Western blots and ELISA assays, and Na+/K+-ATPase activity was evaluated using an ATPase assay in the presence of ouabain. Results show that physiologic pressure and flow increase the transcription and the protein expression of Na+/K+-ATPase α1 in engineered corneal endothelia, while they remain stable in native corneal endothelia. Interestingly, the activity of Na+/K+-ATPase was increased in the presence of physiologic pressure and flow in both native and engineered corneal endothelia. These findings highlight the role of the in vivo environment on the functionality of the corneal endothelium.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration., Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C., Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration., Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

This study describes the development and characterization of novel composite scaffolds, made of an alginate-chitosan hydrogel matrix containing eggshell (ES) particles, for bone tissue engineering applications. Scaffolds with ES particles, either untreated or treated with phosphoric acid to create a nanotextured particle surface, were compared to scaffolds without particles. Results indicate that the nanotexturing process exposed occluded ES proteins orthologous to those in human bone extracellular matrix. Scaffolds with ES or nanotextured ES (NTES) particles had a higher porosity (81 ± 4% and 89 ± 5%, respectively) than scaffolds without particles (59 ± 5%) (p = .002 and p < .001, respectively). Scaffolds with NTES particles had a larger median pore size (113 μm [interquartile range [IQ]: 88-140 μm]) than scaffolds with ES particles (94 μm [IQ: 75-112 μm]) and scaffolds without particles (99 μm [IQ: 74-135 μm]) (p < .001 and p = .011, respectively). The compressive modulus of the scaffolds with ES or NTES particles remained low (3.69 ± 0.70 and 3.14 ± 0.62 kPa, respectively), but these scaffolds were more resistant to deformation following maximum compression than those without particles. Finally, scaffolds with ES or NTES particles allowed better retention of human mesenchymal stem cells during seeding (53 ± 12% and 57 ± 8%, respectively, vs. 17 ± 5% for scaffolds without particles; p < .001 in both cases), as well as higher cell viability up to 21 days of culture (67 ± 17% and 61 ± 11%, respectively, vs. 15 ± 7% for scaffolds without particles; p < .001 in both cases). In addition, alkaline phosphatase (ALP) activity increased up to 558 ± 164% on day 21 in the scaffolds with ES particles, and up to 567 ± 217% on day 14 in the scaffolds with NTES particles (p = .006 and p = .002, respectively, relative to day 0). Overall, this study shows that the physicochemical properties of the alginate-chitosan hydrogel scaffolds with ES or NTES particles are similar to those of cancellous bone. In addition, scaffolds with particles supported early osteogenic differentiation and therefore represent a promising new bone substitute, especially for non-load bearing applications., (© 2023 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Cell monolayers that form a barrier between two structures play an important role for the maintenance of tissue functionality. In the anterior portion of the eye, the corneal endothelium forms a barrier that controls fluid exchange between the aqueous humor of the anterior chamber and the corneal stroma. This monolayer is central in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). FECD is a common corneal disease, in which corneal endothelial cells deposit extracellular matrix that increases the thickness of its basal membrane (Descemet's membrane), and forms excrescences (guttae). With time, there is a decrease in endothelial cell density that generates vision loss. Transplantation of a monolayer of healthy corneal endothelial cells on a Descemet membrane substitute could become an interesting alternative for the treatment of this pathology. In the back of the eye, the retinal pigment epithelium (RPE) forms the blood-retinal barrier, controlling fluid exchange between the choriocapillaris and the photoreceptors of the outer retina. In the retinal disease dry age-related macular degeneration (dry AMD), deposits (drusen) form between the RPE and its basal membrane (Bruch's membrane). These deposits hinder fluid exchange, resulting in progressive RPE cell death, which in turn generates photoreceptor cell death, and vision loss. Transplantation of a RPE monolayer on a Bruch's membrane/choroidal stromal substitute to replace the RPE before photoreceptor cell death could become a treatment alternative for this eye disease. This review will present the different biomaterials that are proposed for the engineering of a monolayer of corneal endothelium for the treatment of FECD, and a RPE monolayer for the treatment of dry AMD., 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 © 2023 Sasseville, Karami, Tchatchouang, Charpentier, Anney, Gobert and Proulx.)

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by an accelerated depletion of corneal endothelial cells. There is growing evidence that mitochondrial exhaustion is central in the pathology. Indeed, endothelial cells loss in FECD forces the remaining cells to increase their mitochondrial activity, leading to mitochondrial exhaustion. This generates oxidation, mitochondrial damage, and apoptosis, fueling a vicious cycle of cells' depletion. This depletion ultimately causes corneal edema and irreversible loss of transparency and vision. Concurrently to endothelial cells loss, the formation of extracellular mass called guttae on the Descemet's membrane, is a hallmark of FECD. The pathology origins at the center of the cornea and progress outward, like the appearance of guttae., Methods: Using corneal endothelial explants from patients with late-stage FECD at the time of their corneal transplantation, we correlated mitochondrial markers (mitochondrial mass, potential, and calcium) and the level of oxidative stress and apoptotic cells, with the area taken by guttae. The different markers have been analyzed using fluorescent-specific probes and microscopic analysis., Results: We observed a positive correlation between the presence of guttae and the level of mitochondrial calcium and apoptotic cells. We found a negative correlation between the presence of guttae and the level of mitochondrial mass, membrane potential, and oxidative stress., Conclusions: Taken together, these results show that the presence of guttae is correlated with negative outcome in the mitochondrial health, oxidative status, and survival of nearby endothelial cells. This study provides insight on FECD etiology that could lead to treatment targeting mitochondrial stress and guttae.

Fuchs endothelial corneal dystrophy (FECD) is characterized by an accelerated loss of corneal endothelial cells. Since the function of these cells is to maintain the cornea in a state of deturgescence necessary for its transparency, the depletion of corneal endothelial cells ultimately causes corneal edema and irreversible loss of vision. Evidence is accumulating regarding the central involvement of mitochondria in FECD. As we have previously shown, when endothelial cells die and are not replaced, the mitochondria of surviving cells must provide more energy to compensate, leading to a phenomenon we have called mitochondrial burnout. This burnout causes cell death, thus exacerbating an irreversible vicious circle responsible for FECD progression. Corneal transplantation, for which the transplant supply is insufficient, is the only curative alternative for FECD. It thus becomes imperative to find other avenues of treatment. In this article, we tested whether incorporating healthy mitochondria into FECD cells would improve pathological molecular markers of the disease. Using corneal endothelium explants from FECD patients, we demonstrated that incorporation of exogenous mitochondria into FECD cells by co-incubation reduces oxidative stress, increases mitochondrial membrane potential, and reduces mitophagy. In addition, internalization of exogenous mitochondria significantly reduces apoptosis (57% in FECD vs 12% in FECD with internalized mitochondria). Taken together, these results suggest that the internalization of exogenous mitochondria reverses the vicious circle involved in FECD, thus revealing a much-needed novel treatment alternative for FECD., (© 2023. The Author(s).)

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, β-catenin, GSK-3β and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Purpose: Transforming growth factor-beta (TGF-β) is known to influence many cell functions. In the corneal endothelium, TGF-β1 exerts contextual effects, promoting endothelial-mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-β isoforms participate in the formation of corneal endothelial intercellular junctions., Methods: Corneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-β isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-β2 were also studied. The beneficial effect of TGF-β2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor., Results: The results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-β isoforms were individually added during the maturation phase, and TGF-β2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-β2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points., Conclusions: These results suggest that TGF-β2 can strengthen cell-cell and cell-substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

Purpose: Tissue engineering of the corneal endothelium, as well as cell therapy, has been proposed as an alternative approach for the treatment of corneal endotheliopathies. These approaches require in vitro amplification of functional corneal endothelial cells (CECs). The goal of this study was to compare two common isolation methods, collagenase A and EDTA (EDTA), and determine whether they influence cell viability, morphology, and barrier function., Methods: Human eye bank research-grade corneas were used to isolate and cultivate CECs. All donors were more than 40 years old. Two Descemet membranes from the same donor were used separately to compare the collagenase A and EDTA cell isolation methods. The number of isolated cells, cell viability, morphology, and barrier functionality were compared., Results: A higher isolation efficiency of viable CECs and a higher circularity index (endothelial morphology) were obtained using collagenase A. Passage 3 cells presented similar barrier functionalities regardless of the isolation method., Conclusions: This study showed that isolation of CECs using collagenase A yields higher isolation efficiency than EDTA, delaying the loss of endothelial morphology for early passage cells., (Copyright © 2022 Molecular Vision.)

Inadequate nutritional rehabilitation of severely malnourished adolescents with Anorexia Nervosa (AN) increases the risk of medical complications. There is no consensus on best practices for inpatient nutritional rehabilitation and medical stabilization for severe AN. This study aimed to elaborate an admission protocol for adolescents with severe AN based on a comprehensive narrative review of current evidence. A Pubmed search was conducted in July 2017 and updated in August 2020, using the keywords severe AN or eating disorders (ED), management guidelines and adolescent. Relevant references cited in these guidelines were retrieved. A secondary search was conducted using AN or ED and refeeding protocol, refeeding syndrome (RS), hypophosphatemia, hypoglycemia, cardiac monitoring or cardiac complications. Evidence obtained was used to develop the admission protocol. Selective blood tests were proposed during the first three days of nutritional rehabilitation. Higher initial caloric intake is supported by evidence. Continuous nasogastric tube feeding was proposed for patients with a BMI < 12 kg/m2. We monitor hypoglycemia for 72 h. Continuous cardiac monitoring for bradycardia <30 BPM and systematic phosphate supplementation should be considered. Developing protocols is necessary to improve standardization of care. We provide an example of an inpatient admission protocol for adolescents with severe AN.

Objective: We determined secular changes in the incidence of hospitalizations due to herpes zoster (HZh) and assessed the validity of HZ International Classification of Diseases (ICD) code algorithms for identifying HZh in a region of Quebec, Canada., Methods: We performed a validation study as part of a retrospective cohort study of adult HZ patients hospitalized at Centre Hospitalier Universitaire de Sherbrooke during 2000-2017. Cases were identified using ICD codes from an inpatient administrative database. HZ cases identified by ICD-9 (053.xx) and ICD-10 (B02.x) codes were chart-confirmed, and performance characteristics of ICD code algorithms were calculated (positive predictive value [PPV] and sensitivity)., Results: Overall, 1314 hospitalizations with HZ diagnosis (HZh) with or without complications were identified during 2000-2017. Among the hospitalizations, 526 (44.4%) were due to active HZ disease or a complication related to a recent or previous HZ episode. These hospitalizations were due to active disease at the time of admission (340/526, 64.6%), HZ that developed during hospitalization (120/526, 22.8%), or a complication directly related to a recent or previous HZ episode (66/526, 12.6%). PPV was significantly higher when HZ was the primary diagnosis (276/310, 89%, 95% confidence interval [CI]: 85-92%) than when HZ was a secondary diagnosis (254/928, 27%, 95% CI: 25-30%) (p < 0.0001), and the PPV of a first secondary diagnosis (84/140, 60.0%, 95% CI: 51.3-68.2%) was higher than that of other secondary diagnoses (203/794, 25.6%, 95% CI: 22.6-28.8%) (p < 0.0001). An algorithm combining ICD codes and antiviral usage demonstrated the best sensitivity (86.3%, 95% CI: 83.1-89.1%) and PPV to identify HZh (100%, 95% CI: 99.2-100%). Poisson regression revealed no significant changes in HZh over time (incidence rate ratio: 0.98, 95% CI: 0.92-1.04%; p = 0.5)., Conclusion: HZh incidence was stable over time. Prescription of antivirals might be a useful addition to ICD codes to identify HZh cases from administrative databases., 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., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Mechanicals forces are known to influence cell behavior. In vivo, the corneal endothelium is under the influence of various mechanical forces, such as intraocular pressure (IOP) and fluid flow. In this study, we used a corneal bioreactor to understand the effect of these hydrodynamic forces on the transcription of intercellular junctions associated genes in the corneal endothelium. Native and tissue-engineered (TE) corneal endothelium were cultured in a corneal bioreactor for 7 days with 16 mmHg IOP and 5 μl/ml of medium flow. RNA was harvested, and gene expression was quantified. Cells that were used to reconstruct the TE corneal endothelia were also seeded on plastic to characterize their morphology by calculating their circularity index. For native endothelia, hydrodynamic forces increased gene expression of GJA1 (connexin 43), CDH2 (N-cadherin), TJP1 (ZO-1), ITGAV (integrin subunit αv), ITGB5 (integrin subunit β5) and CTNND1 (p120-ctn) by 1.68 ± 0.40, 1.10 ± 0.27, 3.80 ± 0.56, 1.82 ± 0.33, 1.32 ± 0.21 and 3.04 ± 0.63, respectively. For TE corneal endothelium, this fold change was 1.72 ± 0.31, 1.58 ± 0.41, 6.18 ± 1.03, 1.80 ± 0.71, 1.77 ± 0.55, 2.42 ± 0.71. Furthermore, gene transcription fold changes (hydrodynamic/control) increased linearly with TE corneal endothelium cells population morphology with r = 0.83 for TJP1 (ZO-1) and r = 0.58 for CTNND1 (p120-ctn). In fact, the more elongated the cells populations were, the greater hydrodynamic conditions increased the transcription of TJP1 (ZO-1) and CTNND1 (p120-ctn). These results suggest that hydrodynamic forces contribute to the maintenance of tight and adherens junctions of native corneal endothelial cells, as well as to the formation of tight and adherens junctions of corneal endothelial cells that are in the process of forming a functional endothelial barrier., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Evaluate the experience of virtual consultations for young people and their families and assess whether young people are being offered a confidential space as part of these virtual encounters., Patients and Methods: An anonymous online survey was sent to young people age 10-18 y.o. who had experienced at least one virtual consultation with an adolescent medicine tertiary service in the United Kingdom between March 13th and June 13th 2020 mostly associated with, but not exclusively, management of chronic fatigue syndrome or medically unexplained symptoms. Responses from the survey were analysed by two authors who independently coded the common themes reported by the participants., Results: Fifty young people and their families participated in the survey. Eighty-eight percent reported feeling prepared for virtual appointments, 90% found them helpful, 88% felt that they were private and 86% reported they would find further virtual appointments helpful. Positive impacts reported were no need to travel (38%) and the continuity of care (36%). Many of our participants reported no negative impact (39%) and felt that nothing needed to be improved (56%). The most frequent improvement reported was the provision of a quality video call (34%). Only 36% of young people had the opportunity to speak in confidence to the health care provider without their parents' presence., Conclusion: Virtual appointments are perceived as safe and helpful by the young people and their families. Professionals should offer a confidential remote space for young people to speak without their parents., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Proulx-Cabana et al.)

Fuchs endothelial corneal dystrophy (FECD) is characterized by a progressive loss of corneal endothelial cells (CECs) and an abnormal accumulation of extracellular matrix in Descemet's membrane leading to increased thickness and formation of excrescences called guttae. Extracellular matrix homeostasis is modulated by an equilibrium between matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). This study aimed to investigate MMPs and TIMPs profile in FECD, taking into account cell morphology. Populations of FECD and healthy CECs were cultured and their conditioned media collected for analysis. The presence of proteases in the conditioned media was studied using a semi-quantitative proteome profiler array, and MMPs levels were assessed using quantitative assays (ELISA and quantitative antibody array). MMP activity was determined by zymography and fluorometry. The expression pattern of the membrane type 1-MMP (MT1-MMP, also known as MMP-14) was examined by immunofluorescence on ex vivo FECD and healthy explants of CECs attached to Descemet's membrane. Finally, MMPs and TIMPs protein expression was compared to gene expression obtained from previously collected data. FECD and healthy CEC populations generated cultures of endothelial, intermediate, and fibroblastic-like morphology. Various MMPs (MMP-1, -2, -3, -7, -8, -9, -10, and -12) and TIMPs (TIMP-1 to -4) were detected in both FECD and healthy CECs culture supernatants. Quantitative assays revealed a decrease in MMP-2 and MMP-10 among FECD samples. Both these MMPs can degrade the main extracellular matrix components forming guttae (fibronectin, laminin, collagen IV). Moreover, MMPs/TIMPs ratio was also decreased among FECD cell populations. Activity assays showed greater MMPs/Pro-MMPs proportions for MMP-2 and MMP-10 in FECD cell populations, although overall activities were similar. Moreover, the analysis according to cell morphology revealed among healthy CECs, both increased (MMP-3 and -13) and decreased (MMP-1, -9, -10, and -12) MMPs proteins along with increased MMPs activity (MMP-2, -3, -9, and -10) in the fibroblastic-like subgroup when compared to the endothelial subgroup. However, FECD CECs did not show similar behaviors between the different morphology subgroups. Immunostaining of MT1-MMP on ex vivo FECD and healthy explants revealed a redistribution of MT1-MMP around guttae in FECD explants. At the transcriptional level, no statistically significant differences were detected, but cultured FECD cells had a 12.2-fold increase in MMP1 and a 4.7-fold increase in TIMP3. These results collectively indicate different, and perhaps pathological, MMPs and TIMPs profile in FECD CECs compared to healthy CECs. This is an important finding suggesting the implication of MMPs and TIMPs in FECD pathophysiology., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Anorexia Nervosa (AN) is a highly prevalent disease in adolescents and young adults (AYAs). The quality of initial inpatient medical management in a patient with severe malnutrition due to AN is crucial to prevent further medical instability. This review aims to inventory evidence regarding initial refeeding and management of AYAs with AN in an inpatient setting, in order to avoid medical complications., Methods: A systematic review will be conducted using PubMed, MEDLINE, All EBM Reviews, Embase, PsycINFO, Cochrane Database and CINAHL. The search strategy consists of terms related to anorexia, hospitalization and Pediatrics. Randomized controlled trials, case-control studies, cross-sectional and cohort studies will be included. Participants will include adolescents and adults 18-24 years old diagnosed with AN and meeting criteria for severe disease. The primary outcome will be any of the following complications: hypophosphatemia, refeeding syndrome, hypoglycemia, cardiac arrythmia, hepatic cytolysis or death. Data will be extracted and the risk of bias will be assessed by one author and reviewed by a second author. Results will be presented in a systematic narrative synthesis format. The quality of evidence for all outcomes will be evaluated using the GRADE methodology., Discussion: This systematic review will examine current evidence on initial inpatient refeeding and help to document effectiveness of initial inpatient management in AYAs with severe AN in avoiding further medical complications., Trial Registration: This study is registered on PROSPERO under the reference number CRD42019123608 .

This study aimed at better understanding the neurochemistry underlying transcranial magnetic stimulation (TMS) and magnetic resonance spectroscopy (MRS) measurements as it pertains to GABAergic activity following administration of allosteric GABA A receptor agonist lorazepam. Seventeen healthy adults (8 females, 26.0 ± 5.4 years old) participated in a double-blind, crossover, placebo-controlled study, where participants underwent TMS and MRS two hours after drug intake (placebo or lorazepam; 2.5 mg). Neuronavigated TMS measures reflecting cortical inhibition and excitation were obtained in the left primary motor cortex. Sensorimotor cortex and occipital cortex MRS data were acquired using a 3T scanner with a MEGA-PRESS sequence, allowing water-referenced [GABA] and [Glx] (glutamate + glutamine) quantification. Lorazepam administration decreased occipital [GABA], decreased motor cortex excitability and increased GABA A -receptor mediated motor cortex inhibition (short intracortical inhibition (SICI)). Lorazepam intake did not modulate sensorimotor [GABA] and TMS measures of intra-cortical facilitation, long-interval cortical inhibition, cortical silent period, and resting motor threshold. Furthermore, higher sensorimotor [GABA] was associated with higher cortical inhibition (SICI) following lorazepam administration, suggesting that baseline sensorimotor [GABA] may be valuable in predicting pharmacological or neuromodulatory treatment response. Finally, the differential effects of lorazepam on MRS and TMS measures, with respect to GABA, support the idea that TMS measures of cortical inhibition reflect synaptic GABAergic phasic inhibitory activity while MRS reflects extrasynaptic GABA., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

There is growing evidence that river temperatures are increasing under climate change, which is expected to be exacerbated by increased abstractions to satisfy human water demands. Water temperature research has experienced crucial advances, both in terms of developing new monitoring and modelling tools, as well as understanding the mechanisms of temperature feedbacks with biogeochemical and ecological processes. However, water practitioners and regulators are challenged with translating the widespread and complex technological, modelling and conceptual advances made in river temperature research into improvements in management practice. This critical review provides a comprehensive overview of recent advances in the state-of-the-art monitoring and modelling tools available to inform ecological research and practice. In so doing, we identify pressing research gaps and suggest paths forward to address practical research and management challenges. The proposed research directions aim to provide new insights into spatio-temporal stream temperature dynamics and unravel drivers and controls of thermal river regimes, including the impacts of changing temperature on metabolism and aquatic biogeochemistry, as well as aquatic organisms. The findings of this review inform future research into ecosystem resilience in the face of thermal degradation and support the development of new management strategies cutting across spatial and temporal scales., 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 © 2020 Elsevier B.V. All rights reserved.)

Fuchs endothelial corneal dystrophy (FECD) is a degenerative eye disease characterized by corneal endothelial cell (CEC) death and the formation of guttae, an abnormal thickening of CEC's basement membrane. At the tissue level, an oxidative stress causing mitochondrial damage and CEC death have been described to explain FECD pathogenesis. At the cellular level, our group has previously observed significant variability in the mitochondrial mass of FECD CECs. This led us to hypothesize that mitochondrial mass variability might play a key role in the chronology of events eventually leading to CEC death in FECD. We thus used different fluorescent markers to assess mitochondrial health and functionality as a function of mitochondrial mass in FECD corneal endothelial explants, namely, intra-mitochondrial calcium, mitochondrial membrane potential, oxidation level and apoptosis. This has led us to describe for the first time a sequence of events leading to what we referred to as a mitochondrial burnout, and which goes as follow. FECD CECs initially compensate for endothelial cell losses by incorporating mitochondrial calcium to help generating more ATP, but this leads to increased oxidation. CECs then resist the sustained need for more ATP by increasing their mitochondrial mass, mitochondrial calcium and mitochondrial membrane potential. At this stage, CECs reach their maximum capacity and start to cope with irreversible oxidative damage, which leads to mitochondrial burnout. This burnout is accompanied by a dissipation of the membrane potential and a release of mitochondrial calcium, which in turn leads to cell death by apoptosis.

The corneal endothelium forms a leaky barrier between the corneal stroma and the aqueous humor of the anterior chamber. This cell monolayer maintains the corneal stroma in a state of relative dehydration, a process called deturgescence, which is required in order to obtain corneal stromal transparency. Endothelial dysfunctions lead to visual impairment that ultimately can only be treated surgically via the corneal transplantation of a functional endothelium. Shortages of corneas suitable for transplantation has motivated research toward new alternatives involving in vitro corneal endothelial cell (CEC) expansion.This chapter describes current methods that allow isolate and culture CECs. In brief, Descemet membrane is peeled out of the cornea and digested in order to obtain CECs. Cells are then seeded and cultured.

The purpose of the present study was to investigate the long-term stability of water-referenced GABA and Glx neurometabolite concentrations in the sensorimotor cortex using MRS and to assess the long-term stability of GABA- and glutamate-related intracortical excitability using transcranial magnetic stimulation (TMS). Healthy individuals underwent two sessions of MRS and TMS at a 3-month interval. A MEGA-PRESS sequence was used at 3 T to acquire MRS signals in the sensorimotor cortex. Metabolites were quantified by basis spectra fitting and metabolite concentrations were derived using unsuppressed water reference scans accounting for relaxation and partial volume effects. TMS was performed using published standards. After performing stability and reliability analyses for MRS and TMS, reliable change indexes were computed for all measures with a statistically significant test-retest correlation. No significant effect of time was found for GABA, Glx and TMS measures. There was an excellent ICC and a strong correlation across time for GABA and Glx. Analysis of TMS measure stability revealed an excellent ICC for rMT CSP and %MSO and a fair ICC for 2 ms SICI. There was no significant correlation between MRS and TMS measures at any time point. This study shows that MRS-GABA and MRS-Glx of the sensorimotor cortex have good stability over a 3-month period, with variability across time comparable to that reported in other brain areas. While resting motor threshold, %MSO and CSP were found to be stable and reliable, other TMS measures had greater variability and lesser reliability.

Production of chemicals in microorganisms is no longer restricted to products arising from native metabolic potential. In this review, we highlight the evolution of metabolic engineering studies, from the production of natural chemicals fermented from biomass hydrolysates, to the engineering of microorganisms for the production of non-natural chemicals. Advances in synthetic biology are accelerating the successful development of microbial cell factories to directly produce value-added chemicals. Here we outline the emergence of novel computational tools for the creation of synthetic pathways, for designing artificial enzymes for non-natural reactions and for re-wiring host metabolism to increase the metabolic flux to products. We also highlight exciting opportunities for applying directed evolution of enzymes, dynamic control of growth and production, growth-coupling strategies as well as decoupled strategies based on orthogonal pathways in the context of non-natural chemicals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A few hours of monocular patching temporarily enhances the deprived eye's contribution to binocular vision, constituting a form of adult brain plasticity. Although the mechanism for this plasticity is currently unknown, several imaging studies present evidence that monocular deprivation achieves its effects by changing excitatory-inhibitory balance in the visual cortex. Much of the past work on adult monocular patching utilized binocular rivalry to quantify the patching-induced shift in perceptual eye dominance, extracting periods of exclusive visibility (in which one eye's signal is suppressed from perception) to assess each eye's contribution to binocular vision while overlooking the occurrence of mixed visibility (in which information from both eyes is combined). In this paper, we discuss two experiments to investigate the effects of short-term monocular occlusion on the relative predominance of mixed and exclusive percepts during binocular rivalry. In addition to the known perceptual eye-dominance shift, we hypothesized patching would also increase the perception of mixtures during rivalry due to deprivation-induced changes in excitatory-inhibitory balance. Our data point to two previously unknown effects of monocular deprivation: (a) a significant increase in the overall fraction and median duration of mixed visibility during rivalry that is detectable up to at least an hour after removing the patch and (b) the overall fraction of superimposition; rather than piecemeal, mixed percepts are specifically enhanced after monocular deprivation. In addition to strengthening the contribution of the deprived eye, our results show that temporary monocular patching enhances the visibility of fused binocular percepts, likely the result of attenuated interocular inhibition.

While public involvement in health policy is gaining traction around the world, deciding whether practitioners of public involvement should encourage participants to deliberate from a personal or a collective perspective remains an object of contention. Drawing on an empirical study, the aim of this article is to generate methodological insights into these two perspectives. Our qualitative analyses illustrate how members of the public contributed differently to deliberations about the value of health innovations by alternatively sharing views as public representatives and as potential users. When engaging as public representatives, participants raised important collective concerns, and, when engaging as potential users, participants brought concrete details and contextual nuances to the group exchanges. Because these perspectives entail different yet mutually challenging ways of appraising health innovations, public engagement practitioners should foster both personal and collective perspectives., (Copyright © 2019 Longwoods Publishing.)

Cells and tissues are influenced by environmental conditions. In vivo, the corneal endothelium is subjected to hydrostatic intraocular pressure (IOP) and to the hydrokinetic pressure of the moving aqueous humor in the anterior chamber. In this paper, we used a corneal bioreactor to recreate the IOP condition and investigated the effect of the in vivo hydrodynamic environment of corneal endothelial cells on the formation of tight junctions. Native ex vivo corneas and engineered corneal endothelia subjected to pressure showed an increase in ZO-1 expression at the cell periphery. Pressure also improved the corneal transparency of engineered and native corneas. Corneal thickness was accordingly reduced from 926 ± 70 μm to 651 ± 70 μm for the engineered corneal endothelium and from 847 ± 27 μm to 571 ± 23 μm for the native endothelium. These results suggest that the hydrodynamic pressure of the anterior chamber is important for the cell junction integrity of the corneal endothelium., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The choroid of the eye is a vascularized and pigmented connective tissue lying between the retina and the sclera. Increasing evidence demonstrates that, beyond supplying nutrients to the outer retina, the different choroidal cells contribute to the retina's homeostasis, especially by paracrine signaling. However, the precise role of each cell type is currently unclear. Here, we developed a choroidal substitute using the self-assembly approach of tissue engineering. Retinal pigment epithelial (RPE) cells, as well as choroidal stromal fibroblasts, vascular endothelial cells and melanocytes, were isolated from human eye bank donor eyes. Fibroblasts were cultured in a medium containing serum and ascorbic acid. After six weeks, cells formed sheets of extracellular matrix (ECM), which were stacked to produce a tissue-engineered choroidal stroma (TECS). These stromal substitutes were then characterized and compared to the native choroid. Their ECM composition (collagens and proteoglycans) and biomechanical properties (ultimate tensile strength, strain and elasticity) were similar. Furthermore, RPE cells, human umbilical vein endothelial cells and choroidal melanocytes successfully repopulated the stromas. Physiological structures were established, such as a confluent monolayer of RPE cells, vascular-like structures and a pigmentation of the stroma. Our TECS thus recaptured the biophysical environment of the native choroid, and can serve as study models to understand the normal interactions between the RPE and choroidal cells, as well as their reciprocal exchanges with the ECM. This will consequently pave the way to derive accurate insight in the pathophysiological mechanisms of diseases affecting the choroid. STATEMENT OF SIGNIFICANCE: The choroid is traditionally known for supplying blood to the avascular outer retina. There has been a renewed attention directed towards the choroid partly due to its implication in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Since AMD involves the dysfunction of the choroid/retinal pigment epithelium (RPE) complex, a three-dimensional (3D) model of RPE comprising the choroid layer is warranted. We used human choroidal cells to engineer a choroidal substitute. Our approach takes advantage of the ability of cells to recreate their own environment, without exogenous materials. Our model could help to better understand the role of each choroidal cell type as well as to advance the development of new therapeutics for AMD., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Fuchs endothelial corneal dystrophy (FECD) is a corneal pathology that affects the endothelial cell's ability to maintain deturgescence, resulting in a progressive loss of corneal transparency. In this study, we investigated the expression of function-related proteins in corneal endothelial cells using FECD or healthy corneal endothelial cells, either in a cell culture two-dimensional model or in an engineered corneal endothelium three-dimensional tissue model. No statistically significant difference in gene regulation was observed for the function-related families ATP1, SLC4, SLC16, AQP, TJP, and CDH between the FECD and the healthy cell models. Similarly, no difference in barrier integrity (transendothelial electrical resistance measurements and permeability assays) was observed in vitro between FECD and healthy cultured cells. Protein expression of the key function-related families was decreased for Na + /K + -ATPase α1 subunit, monocarboxylate transporters 1 and 4 in native ex vivo end-stage FECD specimens, whereas it returned to levels comparable to that of healthy tissues in the engineered FECD model. These results indicate that cell expansion and tissue engineering culture conditions can generate a corneal endothelium from pathologic FECD cells, with levels of function-related proteins similar to that of healthy tissues. Overall, these results explain why it is possible to reform a functional endothelium using corneal endothelial cells isolated from nonfunctional FECD pathologic specimens., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Primary corneal endothelial cell (CEC) cultures and 3D-engineered tissue models were used to study the aberrant deposition of extracellular matrix (ECM) in a vision impairing pathology known as Fuchs endothelial corneal dystrophy (FECD). CECs were isolated from excised Descemet membranes of patients with end-stage FECD. CECs isolated from healthy corneas served as controls. Microarray gene profiling was performed on postconfluent cultures of healthy and FECD cells. Protein expression analyses were conducted on tissue models that were engineered by seeding an endothelium on previously devitalized human stromal carriers. The engineered endothelia were kept in culture for 1-3 weeks to reform the endothelial monolayer. Protein expression of integrin subunits α4, α6, αv, and β1, as well as laminin, type IV collagen, fibronectin, clusterin, and transforming growth factor β-induced protein (TGFβIp) was then assessed by immunofluorescence. Microarray analysis showed nonstatistical twofold downregulation of collagen-coding genes (COL4A4, COL8A2, and COL21A1) and a twofold upregulation of the COL6A1, laminin α3 gene LAMA3, and integrin subunit α10 gene ITGA10 in FECD cells. Fibronectin type III domain containing 4 (FNDC4) and integrin β5 (ITGB5) genes was significantly upregulated in FECD cells. Immunostainings demonstrated that the protein expression of the integrin subunits α4, α6, αv, and β1, type IV collagen, as well as laminin remained similar between native and engineered endothelia. TGFβIp expression was found on the stromal side of both FECD and healthy Descemet's membrane, and only one out of three FECD specimens was positive for the clusterin protein. Interestingly, the ECM protein fibronectin was also found to have a stronger presence on engineered FECD tissues, a result consistent with the native FECD specimens. To conclude, this study allowed to identify fibronectin deposition as one of the first steps in the pathogenesis of FECD, as defined by our engineered tissue model. This opens the way to an entirely new perspective for in vitro pharmacological testing of new therapies for FECD, the leading indication for corneal transplantation in North America.

The objective of the study was to determine whether repetitive hits to the head at a subclinical level are associated with structural and functional brain abnormalities and whether these effects are influenced by high levels of fitness associated with intense physical activity. Seventy-two college students were recruited: 24 nonathletic, 24 athletes practicing a varsity contact sport, and 24 athletes practicing a varsity noncontact sport. They were recruited for a neuropsychological evaluation and a magnetic resonance imaging session that included magnetic resonance spectroscopy of primary motor cortex (M1) and prefrontal cortex and susceptibility-weighted imaging. There was no evidence for reduced cognitive performance or presence of micro bleeds in contact sports athletes. Abnormalities in contact sports athletes were found for myo-inositol concentration (mIns) in M1, where levels were significantly higher compared with noncontact sports athletes ( p  = 0.016) and nonathletes ( p  = 0.029). In prefrontal cortex, glutamate + glutamine (Glx) was significantly reduced in contact sports athletes compared with noncontact sports athletes ( p  = 0.016), and a similar reduction was observed for gamma-aminobutyric acid (GABA) levels ( p  = 0.005). Varsity contact sports are associated with area-specific alterations in mIns concentration in the primary motor cortex. In the prefrontal cortex, high levels of fitness could modulate the effects of head impact exposure on prefrontal metabolite concentration. Indeed, although athletes in contact and noncontact sports show different neurometabolic profiles, they do not differ from sedentary controls.

Human corneal endothelial cells (HCECs) easily become fibroblastic-like when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor β (TGF-β) could be a key factor in this phenomenon; however, TGF-β is also known to maintain the endothelium in a quiescent state in vivo. This work aimed to compare the effects of TGF-β1 on the phenotype of HCECs during the proliferation and maturation phases. Our results show that addition of TGF-β1 during the active proliferation phase produced fibroblastic HCECs and loss of the cell junction markers ZO-1 and n-cadherin, independent from the presence of epidermal growth factor (EGF). By contrast, addition of TGF-β1 in maturation media containing few mitogens led to an endothelial phenotype and functional cell junctions as HCECs developed a high trans-endothelial resistance. Furthermore, addition of AG-1478, an epithelial growth factor receptor inhibitor, enhanced the gain of the endothelial phenotype and cell barrier function. Overall, these results show that TGF-β1 can be used to promote the formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs. A two-phase culture of HCECs using distinct proliferation and maturation media could also be key for developing ideal HCEC culture conditions.

Converging areas of research have implicated glutamate and γ-aminobutyric acid (GABA) as key players in neuronal signalling and other central functions. Further research is needed, however, to identify microstructural and behavioral links to regional variability in levels of these neurometabolites, particularly in the presence of demyelinating disease. Thus, we sought to investigate the extent to which regional glutamate and GABA levels are related to a neuroimaging marker of microstructural damage and to motor and cognitive performance. Twenty-one healthy volunteers and 47 people with multiple sclerosis (all right-handed) participated in this study. Motor and cognitive abilities were assessed with standard tests used in the study of multiple sclerosis. Proton magnetic resonance spectroscopy data were acquired from sensorimotor and parietal regions of the brains' left cerebral hemisphere using a MEGA-PRESS sequence. Our analysis protocol for the spectroscopy data was designed to account for confounding factors that could contaminate the measurement of neurometabolite levels due to disease, such as the macromolecule signal, partial volume effects, and relaxation effects. Glutamate levels in both regions of interest were lower in people with multiple sclerosis. In the sensorimotor (though not the parietal) region, GABA concentration was higher in the multiple sclerosis group compared to controls. Lower magnetization transfer ratio within grey and white matter regions from which spectroscopy data were acquired was linked to neurometabolite levels. When adjusting for age, normalized brain volume, MTR, total N-acetylaspartate level, and glutamate level, significant relationships were found between lower sensorimotor GABA level and worse performance on several tests, including one of upper limb motor function. This work highlights important methodological considerations relevant to analysis of spectroscopy data, particularly in the afflicted human brain. These findings support that regional neurotransmitter levels are linked to local microstructural integrity and specific behavioral abilities that can be affected in diseases such as multiple sclerosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Corneal blindness is a major cause of blindness in the world and corneal transplantation is the only widely accepted treatment to restore sight in these eyes. However, it is becoming increasingly difficult for eye banks to meet the increasing demand for transplantable tissue, which is in part due to population aging. Donor tissue shortage is therefore a growing concern globally and there is a need for alternatives to human donor corneas. Biosynthetic corneal substitutes offer several significant advantages over native corneas: Large-scale production offers a powerful potential solution to the severe shortage of human donor corneas worldwide; Good manufacturing practices ensure sterility and quality control; Acellular corneal substitutes circumvent immune rejection induced by allogeneic cells; Optical and biomechanical properties of the implants can be adapted to the clinical need; and finally these corneal substitutes could benefit from new advances in biomaterials science, such as surface coating, functionalization and nanoparticles. This review highlights critical contributions from laboratories working on corneal stromal substitutes. It focuses on synthetic inert prostheses (keratoprostheses), acellular scaffolds with and without enhancement of endogenous regeneration, and cell-based replacements. Accent is put on the physical properties and biocompatibility of these biomaterials, on the functional and clinical outcome once transplanted in vivo in animal or human eyes, as well as on the main challenges of corneal stromal replacement. Regulatory and economic aspects are also discussed. All of these perspectives combined highlight the founding principles of the clinical application of corneal stromal replacement, a concept that has now become reality., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

YbeY is part of a core set of RNases in Escherichia coli and other bacteria. This highly conserved endoribonuclease has been implicated in several important processes such as 16S rRNA 3' end maturation, 70S ribosome quality control, and regulation of mRNAs and small noncoding RNAs, thereby affecting cellular viability, stress tolerance, and pathogenic and symbiotic behavior of bacteria. Thus, YbeY likely interacts with numerous protein or RNA partners that are involved in various aspects of cellular physiology. Using a bacterial two-hybrid system, we identified several proteins that interact with YbeY, including ribosomal protein S11, the ribosome-associated GTPases Era and Der, YbeZ, and SpoT. In particular, the interaction of YbeY with S11 and Era provides insight into YbeY's involvement in the 16S rRNA maturation process. The three-way association between YbeY, S11, and Era suggests that YbeY is recruited to the ribosome where it could cleave the 17S rRNA precursor endonucleolytically at or near the 3' end maturation site. Analysis of YbeY missense mutants shows that a highly conserved beta-sheet in YbeY-and not amino acids known to be important for YbeY's RNase activity-functions as the interface between YbeY and S11. This protein-interacting interface of YbeY is needed for correct rRNA maturation and stress regulation, as missense mutants show significant phenotypic defects. Additionally, structure-based in silico prediction of putative interactions between YbeY and the Era-30S complex through protein docking agrees well with the in vivo results., Importance: Ribosomes are ribonucleoprotein complexes responsible for a key cellular function, protein synthesis. Their assembly is a highly coordinated process of RNA cleavage, RNA posttranscriptional modification, RNA conformational changes, and protein-binding events. Many open questions remain after almost 5 decades of study, including which RNase is responsible for final processing of the 16S rRNA 3' end. The highly conserved RNase YbeY, belonging to a core set of RNases essential in many bacteria, was previously shown to participate in 16S rRNA processing and ribosome quality control. However, detailed mechanistic insight into YbeY's ribosome-associated function has remained elusive. This work provides the first evidence that YbeY is recruited to the ribosome through interaction with proteins involved in ribosome biogenesis (i.e., ribosomal protein S11, Era). In addition, we identified key residues of YbeY involved in the interaction with S11 and propose a possible binding mode of YbeY to the ribosome using in silico docking., (Copyright © 2016 Vercruysse et al.)

Purpose: Fuchs' endothelial corneal dystrophy (FECD), a degenerative disease of the corneal endothelium that leads to vision loss, is a leading cause of corneal transplantation. The cause of this disease is still unknown, but the implication of oxidative stress is strongly suggested. In this study, we analyzed the impact of FECD on mitochondrial DNA (mtDNA) integrity and telomere length, both of which are affected by the oxidative status of the cell., Methods: We compared the levels of total mtDNA, mtDNA common deletion (4977 bp), and relative telomere length in the corneal endothelial cells of fresh Descemet's membrane-endothelium explants and cultured cells from healthy and late stage FECD subjects. Oxidant-antioxidant gene expression and sensitivity to ultraviolet A (UVA)- and H2O2-induced cell death were assessed in cultured cells., Results: Our results revealed increased mtDNA levels and telomere shortening in FECD explants. We also found that cell culture restores a normal phenotype in terms of mtDNA levels, telomere length, oxidant-antioxidant gene expression balance, and sensitivity to oxidative stress-induced cell death in the FECD cells compared with the healthy cells., Conclusions: Taken together, these results bring new evidence of the implication of oxidative stress in FECD. They also show that FECD does not evenly affect the integrity of corneal endothelial cells and that cell culture can rehabilitate the molecular phenotypes related to oxidative stress by selecting the more functional FECD cells.

Purpose: The aim of the present study was to assess, in healthy individuals, the impact of M1-M1 tDCS on primary motor cortex excitability using transcranial magnetic stimulation and sensorimotor metabolite concentration using 1H-MRS., Methods: For both experiments, each participant received the three following interventions (20 min tDCS, 1 mA): left-anodal/right-cathodal, left-cathodal/right-anodal, sham. The effects of tDCS were assessed via motor evoked potentials (experiment 1) and metabolite concentrations (experiment 2) immediately after and 12 minutes following the end of stimulation and compared to baseline measurement., Results: No effect of M1-M1 tDCS on corticospinal excitability was found. Similarly, M1-M1 tDCS did not significantly modulate metabolite concentrations. High inter-subject variability was noted. Response rate analysis showed a tendency towards inhibition following left-anodal/right-cathodal tDCS in 50% of participants and increased GABA levels in 45% of participants., Conclusion: In line with recent studies showing important inter-subject variability following M1-supraorbital tDCS, the present data show that M1-M1 stimulation is also associated with large response variability. The absence of significant effects suggests that current measures may lack sensitivity to assess changes in M1 neurophysiology and metabolism associated with M1-M1 tDCS.

Endothelial dysfunctions are the first indication for allogeneic corneal transplantation. Development of a tissue-engineered posterior cornea could be an alternative to the use of native allogeneic tissues. In this paper, we used the self-assembly approach to form a cellularized stromal substitute that served as a carrier for the engineering of an endothelium. This endothelialized stromal substitute was then characterized using alizarin red staining, histology, scanning and transmission electron microscopy, as well as mass spectrometry and immunodetection of collagens and function-related proteins. We report the engineering of a monolayer of flattened endothelial cells with a cell density of 966 ± 242 cells/mm(2) (mean ± SD). Endothelial interdigitations were present between cells. The stromal fibroblasts deposited a dense and cohesive collagenous matrix. Collagen fibrils had a diameter of 39.1 ± 11.3 nm, and a mean center to center interfibrillar space of 50.9 ± 10.9 nm. The stromal substitute was composed of collagen types I, V, VI and XII, as well as lumican and decorin. Type IV collagen was also present underneath the endothelium. The endothelium expressed both the sodium/potassium (Na(+)/K(-)) ATPase and sodium/bicarbonate (Na(+)/ [Formula: see text] ) cotransporter pumps. These results indicate that the self-assembled stromal substitute is able to support the expression of endothelial cell functionality markers and therefore, is a suitable carrier for the engineering of an endothelium that could be used for the treatment of endothelial dysfunctions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the functionality of a corneal endothelium reconstituted by injection of corneal endothelial cells (CEC) in the anterior chamber of a feline model., Methods: We operated the right eyes of 16 animals. Eight underwent central endothelial scraping and injection with 2 × 10(5) (n = 4) or 1 × 10(6) (n = 4) feline CEC supplemented with Y-27632 and labeled with 3,3'-Dioctadecyl-5,5'-Di(4-Sulfophenyl)Oxacarbocyanine (SP-DiOC18[3] or DiOC). After total endothelial scraping, two eyes were injected with 1 × 10(6) labeled CEC and Y-27632. The central (n = 3) or entire (n = 3) endothelium was scraped in six eyes followed by Y-27632 injection without CEC. Subjects were positioned eyes down for 3 hours. Outcomes included graft transparency, pachymetry, CEC morphometry, histology, electron microscopy, and function and wound healing-related protein immunostaining., Results: Postoperatively, corneas grafted with 2 × 10(5) CEC and centrally scraped controls displayed the best transparency and pachymetry. Corneas grafted with 1 × 10(6) CEC yielded intermediate results. Entirely scraped controls remained hazy and thick. Histopathology revealed a confluent endothelial monolayer expressing sodium-potassium adenosine triphosphatase (Na(+)/K(+)-ATPase) and zonula occludens-1 (ZO-1) in corneas grafted with 2 × 10(5) CEC and centrally scraped controls, a nonuniform endothelial multilayer without expression of functional proteins in centrally scraped corneas grafted with 1 × 10(6) CEC, and a nonfunctional fibrotic endothelium in entirely scraped grafts and controls. Expression of DiOC in grafts was scarce., Conclusions: Injected CEC contributed little to the incompletely functional endothelium of grafted corneas. Y-27632 injection without CEC following scraping reconstituted the healthiest endothelium. Further studies investigating the therapeutic effect of Y-27632 alone are needed to validate these conclusions.

Unlabelled: As many as 98,000 people die every year from preventable medical errors. Among pharmacists, the most common error reported is the selection of the wrong drug. Merck met with the U.S. Food and Drug Administration (FDA) to discuss the optimization of the U.S. label for solid oral dosage forms of Merck medications. These discussions led to the development of revised label designs for six products that were then evaluated using failure modes and effects analysis and an expert review by human factors specialists. There were no errors among 425 filled prescriptions in the validation test of the final label. Key changes to the original labels include the use of a non-branded logo, high-contrast color bands for dosage strength, and an enhanced three-dimensional tablet image. The redesigned labels were approved by the US FDA in June 2011., Practical Applications: The redesigned label should improve the accurate selection of medications from pharmacy shelves., (Copyright © 2015 Elsevier Ltd and National Safety Council. All rights reserved.)

EEG-fMRI is an established technique to allow mapping BOLD changes in response to interictal discharges recorded in the EEG of epilepsy patients. Traditional fMRI experiments rely on an echo planar imaging (EPI) sequence with a time resolution given by its time-to-repetition (TR) of ~2 s. Recently, multiple fast fMRI sequences have been developed to get around the limited temporal resolution of the EPI sequence, and achieved a TR in the 100 ms range or lower. One such sequence is called Magnetic Resonance EncephaloGraphy (MREG). Its high temporal resolution should offer increased detection sensitivity and statistical power in the context of epilepsy studies and in fMRI experiments in general. The aim of this work was to investigate the advantages and disadvantages offered by MREG. This was done by superimposing artificial event-related BOLD responses on EPI and MREG background signals, from 5 epileptic patients, that were free of epileptic discharges (spikes) on simultaneously recorded EEG. These functional datasets simulated different spiking rates and hemodynamic response amplitudes, and were analyzed with the commonly used General Linear Model (GLM) with the canonical hemodynamic response function (HRF) as a fixed model of the response. Robustness to violation of the assumptions of the GLM was additionally assessed with similar simulations using variable spike-to-spike response amplitudes and 8 non-canonical HRFs. Consistent with previous work, MREG yields higher maximum statistical t-values than EPI, but our simulations showed these statistics to be inflated, as the false positive rate at a standard threshold was high. At thresholds set to appropriately control specificity, EPI showed better true positive rate and larger cluster size than MREG. However, the lack of an appropriate calibration of the amplitude of the responses across the sequences precludes definitive judgment on their relative sensitivity. In addition, we show that a mismatch between the assumed and actual HRF impairs more MREG detection performance, but that EPI is more affected by non-modeled spike-to-spike variations of response amplitude. Filtering-out physiological noise, which is not aliased at the fast sampling rate of MREG, and the modeling of temporal autocorrelation are advantageous in increasing the detection power of MREG. This simulation study 1) warrants care when interpreting statistical t-values from fast fMRI sequences, 2) proposes thresholds for valid inferences and processing methods for maximal sensitivities, and 3) demonstrates the relative robustness/susceptibility of MREG and EPI to violation of the GLM's assumptions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Noninvasive, real-time pharmacokinetic (PK) monitoring of ketamine, propofol, and valproic acid, and their metabolites was achieved in mice, using secondary electrospray ionization and high-resolution mass spectrometry. The PK profile of a drug influences its efficacy and toxicity because it determines exposure time and levels. The antidepressant and anaesthetic ketamine (Ket) and four Ket metabolites were studied in detail and their PK was simultaneously determined following application of different sub-anaesthetic doses of Ket. Bioavailability after oral administration vs. intraperitoneal injection was also investigated. In contrast to conventional studies that require many animals to be sacrificed even for low-resolution PK curves, this novel approach yields real-time PK curves with a hitherto unmatched time resolution (10 s), and none of the animals has to be sacrificed. This thus represents a major step forward not only in animal welfare, but also major cost and time savings., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three-dimensional tissues, such as the cornea, are now being engineered as substitutes for the rehabilitation of vision in patients with blinding corneal diseases. Engineering of tissues for translational purposes requires a non-invasive monitoring to control the quality of the resulting biomaterial. Unfortunately, most current methods still imply invasive steps, such as fixation and staining, to clearly observe the tissue-engineered cornea, a transparent tissue with weak natural contrast. Second- and third-harmonic generation imaging are well known to provide high-contrast, high spatial resolution images of such tissues, by taking advantage of the endogenous contrast agents of the tissue itself. In this article, we imaged tissue-engineered corneal substitutes using both harmonic microscopy and classic histopathology techniques. We demonstrate that second- and third-harmonic imaging can non-invasively provide important information regarding the quality and the integrity of these partial-thickness posterior corneal substitutes (observation of collagen network, fibroblasts and endothelial cells). These two nonlinear imaging modalities offer the new opportunity of monitoring the engineered corneas during the entire process of production.

Corneal endothelial cells often adopt a fibroblastic-like morphology in culture, a process that has been attributed to epithelial- or endothelial-to-mesenchymal transition (EMT or EndMT). Although being extensively studied in other cell types, this transition is less well characterized in the corneal endothelium. Because of their neuroectodermal origin and their in vivo mitotic arrest, corneal endothelial cells represent a particular tissue that deserves more attention. This review article presents the basic principles underlying EMT/EndMT, with emphasis on the current knowledge regarding the corneal endothelium. Furthermore, this review discusses cell culture conditions and major cell signaling pathways that have been identified as EndMT-triggering factors. Finally, it summarizes strategies that have been developed to inhibit EndMT in corneal endothelial cell culture. The review of current studies on corneal and classical EndMT highlights some research avenues to pursue in the future and underscores the need to extend our knowledge of this process in order to optimize usage of these cells in regenerative medicine., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)