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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.

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.

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.)

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 .

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.

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.)

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.)

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.

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.)

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.

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: 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.)

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.)

Background: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and DNA repair. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase ortholog also exhibits a shorter life span, which can be rescued by vitamin C. In this study, we analyzed the impact of a mutation in the wrn-1 gene and the dietary supplementation of vitamin C on the global mRNA expression of the whole C. elegans by the RNA-seq technology., Results: Vitamin C increased the mean life span of the wrn-1(gk99) mutant and the N2 wild type strains at 25°C. However, the alteration of gene expression by vitamin C is different between wrn-1(gk99) and wild type strains. We observed alteration in the expression of 1522 genes in wrn-1(gk99) worms compared to wild type animals. Such genes significantly affected the metabolism of lipid, cellular ketone, organic acid, and carboxylic acids. Vitamin C, in return, altered the expression of genes in wrn-1(gk99) worms involved in locomotion and anatomical structure development. Proteolysis was the only biological process significantly affected by vitamin C in wild type worms., Conclusions: Expression profiling of wrn-1(gk99) worms revealed a very different response to the addition of vitamin C compared to wild type worms. Finally, vitamin C extended the life span of wrn-1(gk99) animals by altering biological processes involved mainly in locomotion and anatomical structure development.

Purpose: Corneal tissue shortage has become a major concern worldwide, which has motivated the search for alternative solutions to eye bank human eyes for corneal transplantation. Minimally invasive lamellar transplantation and tissue engineering may offer new opportunities for the rehabilitation of diseased corneas. The aim of this study was to evaluate the biocompatibility and functionality of stromal lamellar grafts tissue-engineered (TE) in vitro and transplanted in vivo in the cornea of a feline model., Methods: The corneal stromas were engineered in culture from corneal stromal cells using the self-assembly approach, without the addition of exogenous material or scaffold. Eight healthy animals underwent two intrastromal grafts in one eye and the contralateral eye was used as a control. Animals were followed with slit-lamp ophthalmic examination, corneal esthesiometry and optical coherent tomography. Confocal microscopy, immunofluorescence, histology, and transmission electron microscopy (TEM) were performed at 4 months., Results: Four months after transplantation, the TE-stromal grafts were transparent, functional, and well tolerated by the eye. All grafts remained avascular, with no signs of immune rejection, despite a short course of low-dose topical steroids. Corneal sensitivity returned to preoperative level and reinnervation of the grafts was confirmed by confocal microscopy and immunofluorescence. Histology and TEM of the TE-grafts showed a lamellar stromal structure with regular collagen fibril arrangement., Conclusions: These results open the way to an entirely new therapeutic modality. Intracorneal filling using a biocompatible, transparent, and malleable TE-stroma could be the basis for multiple types of novel therapeutic options in corneal interventional surgery., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Clinical evidence and structural neuroimaging studies linked cerebellar deficits to cognitive-related symptoms in schizophrenia. Yet, in functional neuroimaging literature to date, the role of the cerebellum in schizophrenia was not explored in a systematic fashion. Here, we reviewed 234 functional magnetic resonance imaging studies indexed by PubMed and published in 1997-2010 that had at least one group of schizophrenia patients, used blood oxygenation level dependent contrast and the general linear model to assess neuronal activity. We quantified presence/absence of cerebellar findings and the frequency of hypo- and hyperactivations (ie, less or more activity in patients relative to healthy controls). We used peaks of activations reported in these studies to build a topographical representation of group differences on a cerebellar map. Cerebellar activity was reported in patients in 41.02% of the articles, with more than 80% of these dedicated to cognitive, emotional, and executive processes in schizophrenia. Almost two-thirds of group comparisons resulted in cerebellar hypoactivation, with a frequency that presented an inverted U shape across different age categories. The majority of the hypoactivation foci were located in the medial portion of the anterior lobe and the lateral hemispheres (lobules IV-V) of the cerebellum. Even though most experimental manipulations did not target explicitly the cerebellum's functions in schizophrenia, the cerebellar findings are frequent and cerebellar hypoactivations predominant. Therefore, although the cerebellum seems to play an important functional role in schizophrenia, the lack of reporting and interpretation of these data may hamper the full understanding of the disorder.

Transcranial magnetic stimulation (TMS) can provide an index of intracortical excitability/inhibition balance. However, the neurochemical substrate of these measures remains unclear. Pharmacological studies suggest the involvement of GABAA and GABAB receptors in TMS protocols aimed at measuring intracortical inhibition, but this link remains inferential. Proton magnetic resonance spectroscopy ((1)H-MRS) permits measurement of GABA and glutamate + glutamine (Glx) concentrations in the human brain and might help in the direct empirical assessment of the relationship between TMS inhibitory measures and neurotransmitter concentrations. In the present study, MRS-derived relative concentrations of GABA and Glx measured in the left M1 of healthy participants were correlated with TMS measures of intracortical inhibition. Glx levels were found to correlate positively with TMS-induced silent period duration, whereas no correlation was found between GABA concentration and TMS measures. The present data demonstrate that specific TMS measures of intracortical inhibition are linked to shifts in cortical Glx, rather than GABA neurotransmitter levels. Glutamate might specifically interact with GABAB receptors, where higher MRS-derived Glx concentrations seem to be linked to higher levels of receptor activity.

Purpose: We evaluated the in vivo functionality of a corneal endothelium tissue-engineered using corneal endothelial cells from human patients with Fuchs endothelial corneal dystrophy (FECD)., Methods: A total of 15 healthy cats underwent full-thickness corneal transplantation. All transplants were of xenogeneic human origin and all grafts but two were tissue-engineered. In seven animals the graft corneal endothelium was tissue-engineered using cultured corneal endothelial cells from humans with FECD (TE-FECD). Two control animals were grafted with an endothelium engineered using cultured endothelial cells from normal eye bank corneas (TE-normal). Two controls received a native full-thickness corneal transplant, and four other controls were grafted with the stromal carrier only (without endothelial cells). Outcome parameters included graft transparency (0, opaque to 4, clear), pachymetry, optical coherence tomography, endothelial cell morphometry, transmission electron microscopy (TEM), and immunostaining of function-related proteins., Results: Seven days after transplantation, 6 of 7 TE-FECD grafts, all TE-normal grafts, and all normal native grafts were clear (transparency score >3), while all carriers-only grafts were opaque (score <1). The mean pachymetry was 772 ± 102 μm for TE-FECD, 524 ± 11 μm for TE-normal, 555 ± 48 for normal native, and 1188 ± 223 μm for carriers only. TEM showed subendothelial loose fibrillar material deposition in all TE-FECD grafts. The TE endothelium expressed Na(+)-K(+)/ATPase and Na(+)/HCO3(-)., Conclusions: Restoration of transparency and corneal thickness demonstrated that the TE-FECD grafts were functional in vivo. This novel FECD seven-day living model suggests a potential role for tissue engineering leading to FECD cell rehabilitation.

Gene duplication can increase an organism's ability to mask the effect of deleterious alleles present in the population, but this is typically a small effect when the source of the genetic variation is mutation. Migration can introduce orders of magnitude more deleterious alleles per generation and may therefore be an important force acting on the structure of genomes. Using formal analytical methods, we study the invasion of haplotypes containing two copies of the resident allele, assuming that a single-locus equilibrium is already established in a continent-island model of migration. Provided that the immigrant allele can be completely masked by multiple functional gene copies, a new duplication will deterministically spread so long as duplicate haplotypes are, on average, fitter than single-copy haplotypes. When fitness depends on the number of immigrant allele copies and their masking ability then the threshold for invasion depends on the rate of immigration and the rate of recombination between the gene copies. Results from several special cases, including formation of protein dimers and Dobzhansky-Muller incompatibilities, suggest that duplications can invade in a wide range of selection regimes. We hypothesize that duplication in response to gene flow may provide an explanation for the high levels of polymorphism in gene copy number observed in natural populations., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

Purpose: The authors conducted in vivo assessment of corneal endothelial toxicity of air and SF6 in the feline model. This research was motivated by the increased use of air in anterior segment surgery in human subjects., Methods: This was a prospective masked study. The eyes of 16 healthy adult cats were randomly assigned for the injection of 0.7 mL air into the anterior chamber of one eye and SF6 in the contralateral eye. Daily examination included slit lamp photographs, pachymetry, and tonometry. Specular microscopy was performed before, 7 days after, and 10 days after injection. The animals were euthanatized, and the corneas were processed for alizarin red-trypan blue staining and for light and electron microscopy., Results: SF6 remained in the anterior chamber significantly longer than air. Both groups showed postinjection inflammation, which on average was maximal at day 2 and more severe with SF6. No difference in IOP was observed between the two groups. Specular microscopy showed significant endothelial cell loss in the SF6 group (mean postinjection cell loss, 132 ± 50 cells/mm(2)) but not in the group injected with air. Alizarin red staining revealed significant regional differences in cell density only in the SF6 group and more pronounced endothelial cell loss in the superior area., Conclusions: These results indicate that both air and SF6 injected into the anterior chamber of the eye can induce intraocular reaction in the feline model and that SF6 is more toxic than air in terms of endothelial cell loss and anterior chamber inflammation.

This review presents the results of studies carried out in our laboratory that aim to investigate, through functional magnetic resonance imaging (fMRI), the brain plasticity associated with motor sequence learning, defined as our ability to integrate simple stereotyped movements into a single motor representation. Following a brief description of Doyon and colleagues' model (2002, 2005, 2009) of motor skill learning that has guided this work, we then describe the functional changes that occur at the different (rapid, slow, automatization) acquisition phases, and propose specific roles that the putamen, the cerebellum and their motor-related cortical areas, play in this form of motor behavior. Finally, we put forward evidence that post-training, non-REM sleep (and spindles in Stage 2 sleep, in particular) contributes to the consolidation of a motor sequence memory trace, and that increased activity within the striatum and/or the hippocampus mediates this mnemonic process.

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour.

A pivotal role in guiding mesenchymal stem cell (MSC) differentiation has recently been attributed to the primary cilium. This solitary, non-motile microtubule-based organelle emerging from the cell surface acts as a sensorial membrane structure reflecting developmental and adaptive processes associated with pathologies including human cystic kidney disease, skeletal malformations, obesity and cancer. Given that the intrinsic hypoxic adaptation of MSC remains poorly understood within ischemic tissues or hypoxic tumours, we questioned whether the hypoxia inducible factor-1α (HIF-1α) might be a downstream effector regulating cilium maintenance. We show that murine bone marrow-derived MSC cultured under hypoxic conditions (1.2% O(2)) lose their primary cilia in a time-dependent manner. Gene silencing of HIF-1α prevented cilia loss in hypoxic cultures, and generation of MSC expressing a constitutively active HIF-1α (MSC-HIF) was found to decrease primary cilium formation. A Wnt pathway-related gene expression array was also performed on MSC-HIF and indicated that the secreted Frizzled-related proteins (sFRP)-1, -3 and -4 were down-regulated, while sFRP-2 was up-regulated. Overexpression of recombinant sFRP-2 or gene silencing of sFRP-1, -3 and -4 in MSC led to primary cilium disruption. These results indicate a molecular signalling mechanism for the hypoxic disruption of the primary cilium in MSC involving an HIF-1α/sFRP axis. This mechanism contributes to our understanding of the adaptive processes possibly involved in the oncogenic transformation and tumour-supporting potential of MSC. Our current observations also open up the possibility for the primary cilia to serve as a biomarker in MSC adaptation to low oxygen tension within (patho)physiological microenvironments.

Purpose: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach., Methods: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed., Results: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1., Conclusions: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP's catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-kappaB) p65(-/-) mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-kappaB1 (p50)(-/-) and in I kappaB kinase (IKK) gamma(-/-) mutant MEF cells. Collectively, our results highlight an IKK/NF-kappaB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-kappaB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells.

Purpose: To evaluate the functional outcome of tissue-engineered corneal endothelium reconstructed on a devitalized carrier and transplanted in the living feline model., Methods: Eighteen healthy adult cats underwent full-thickness corneal transplantation. In 11 animals, the donor cornea was reconstructed from cultured allogeneic feline corneal endothelial cells seeded on the denuded Descemet's membrane of a devitalized human cornea. The reconstructed corneal endothelium was cultured for 2 weeks before transplantation. Five control animals received autologous (n = 1), allogeneic (n = 3), or human xenogeneic (n = 1) native cornea. Two other control animals were grafted with the devitalized carrier only (no cells). Animals were observed daily by slit lamp until euthanatization on day 7. Postmortem analysis included optical coherence tomography (OCT), alizarin red staining, histology, fluorescence microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM)., Results: Nine of the 11 reconstructed corneal endothelial grafts and all five native (autologous, allogeneic, xenogeneic) control grafts were clear and thin 7 days after grafting. In contrast, the two control grafts consisting of the carrier only (without endothelium) remained thick and opaque. Alizarin red staining, histology, SEM, and TEM showed that the transplanted reconstructed endothelium maintained a normal morphology and ultrastructure and expressed the function-related proteins Na(+)/K(+)-ATPase alpha1, Na(+)/HCO(3), and ZO-1., Conclusions: This study provides evidence for the short-term (7-day) anatomic and functional success of corneal transplantation with a tissue-engineered corneal endothelium reconstructed on a devitalized carrier.

Background: This study aimed at evaluating face and content validity, feasibility and reliability of process quality indicators developed previously in the United States or other countries. The indicators can be used to evaluate care and services for vulnerable older adults affected by cognitive impairment or dementia within an integrated service system in Quebec, Canada., Methods: A total of 33 clinical experts from three major urban centres in Quebec formed a panel representing two medical specialties (family medicine, geriatrics) and seven health or social services specialties (nursing, occupational therapy, psychology, neuropsychology, pharmacy, nutrition, social work), from primary or secondary levels of care, including long-term care. A modified version of the RAND(R)/University of California at Los Angeles (UCLA) appropriateness method, a two-round Delphi panel, was used to assess face and content validity of process quality indicators. The appropriateness of indicators was evaluated according to a) agreement of the panel with three criteria, defined as a median rating of 7-9 on a nine-point rating scale, and b) agreement among panellists, judged by the statistical measure of the interpercentile range adjusted for symmetry. Feasibility of quality assessment and reliability of appropriate indicators were then evaluated within a pilot study on 29 patients affected by cognitive impairment or dementia. For measurable indicators the inter-observer reliability was calculated with the Kappa statistic., Results: Initially, 82 indicators for care of vulnerable older adults with cognitive impairment or dementia were submitted to the panellists. Of those, 72 (88%) were accepted after two rounds. Among 29 patients for whom medical files of the preceding two years were evaluated, 63 (88%) of these indicators were considered applicable at least once, for at least one patient. Only 22 indicators were considered applicable at least once for ten or more out of 29 patients. Four indicators could be measured with the help of a validated questionnaire on patient satisfaction. Inter-observer reliability was moderate (Kappa = 0.57)., Conclusion: A multidisciplinary panel of experts judged a large majority of the initial indicators valid for use in integrated care systems for vulnerable older adults in Quebec, Canada. Most of these indicators can be measured using patient files or patient or caregiver interviews and reliability of assessment from patient-files is moderate.

The preferred body temperature of ectotherms is typically inferred from the observed distribution of body temperatures in a laboratory thermal gradient. For very small organisms, however, that observed distribution might misrepresent true thermal preferences. Tiny ectotherms have limited thermal inertia, and so their body temperature and speed of movement will vary with their position along the gradient. In order to separate the direct effects of body temperature on movement from actual preference behaviour on a thermal gradient, we generate a null model (i.e. of non-thermoregulating individuals) of the spatial distribution of ectotherms on a thermal gradient and test the model using parameter values estimated from the movement of nematodes (Caenorhabditis elegans) at fixed temperatures and on a thermal gradient. We show that the standard lab strain N2, which is widely used in thermal gradient studies, avoids high temperature but otherwise does not exhibit a clear thermal preference, whereas the Hawaiian natural isolate CB4856 shows a clear preference for cool temperatures ( approximately 17 degrees C). These differences are not influenced substantially by changes in the starting position of worms in the gradient, the natal temperature of individuals or the presence and physiological state of bacterial food. These results demonstrate the value of an explicit null model of thermal effects and highlight problems in the standard model of C. elegans thermotaxis, showing the value of using natural isolates for tests of complex natural behaviours.

Purpose: To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology., Methods: PCEC cultures were seeded at an initial cell density of 400 cells/cm(2) in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm(2) in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm(2) in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 microg/ml), ascorbic acid (10, 20, 40 microg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology., Results: Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium., Conclusions: Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells.

Purpose: The retinal pigment epithelium (RPE) is differentiated and mitotically inactive in the normal eye, but several pathologies such as proliferative vitreoretinopathy (PVR) cause RPE cells to dedifferentiate and resume proliferation. Integrins, a family of cell surface glycoproteins that mediate cell proliferation and differentiation, are thought to play fundamental roles in PVR. The aim of this study was to evaluate protein expression and gene regulation of the integrin alpha5 subunit in proliferating and quiescent RPE cells., Methods: Protein expression was studied in situ by immunohistochemistry and in vitro at different cell confluences by immunoprecipitation. Semi-quantitative RT-PCR and transient transfections were used to determine whether increasing cell confluence also affected alpha5 subunit mRNA levels and promoter activity, respectively., Results: We demonstrated that the integrin alpha5 subunit is present at the RPE cell surface both in situ and in vitro, and that alpha5 protein level is influenced by confluence. Levels of integrin alpha5 transcripts are similar for sub-confluent and confluent cells, and a small increase in the promoter activity was observed between sub-confluent and confluent cells. However, both the integrin alpha5 subunit transcript and the alpha5 promoter activity decreased when cells reached post-confluence., Conclusions: We demonstrated that cell confluence affected protein and gene expression of the integrin alpha5 subunit. Proliferating RPE cells expressed high levels of both the alpha5 protein and mRNA transcripts and showed a high promoter activity. However, when cells reached quiescence, alpha5 gene expression was substantially reduced and RPE cells expressed little alpha5 protein at their cell surface.