Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty

PURPOSE: To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. METHODS: DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface. RESULTS: Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04). CONCLUSIONS: Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group

Organ culture storage of pre-prepared corneal donor material for Descemet's membrane endothelial keratoplasty

Purpose To evaluate the effect of media composition and storage method on pre-prepared Descemet's membrane endothelial keratoplasty (DMEK) grafts. Methods 50 corneas were used. Endothelial wound healing and proliferation in different media were assessed using a standard injury model. DMEK grafts were stored using three methods: peeling with free scroll storage; partial peeling with storage on the stroma and fluid bubble separation with storage on the stroma. Endothelial cell (EC) phenotype and the extent of endothelial overgrowth were examined. Global cell viability was assessed for storage methods that maintained a normal cell phenotype. Results 1 mm wounds healed within 4 days. Enhanced media did not increase EC proliferation but may have increased EC migration into the wounded area. Grafts that had been trephined showed evidence of EC overgrowth, whereas preservation of a physical barrier in the bubble group prevented this. In grafts stored in enhanced media or reapposed to the stroma after trephination, endothelial migration occurred sooner and cells underwent endothelial-mesenchymal transformation. Ongoing cell loss, with new patterns of cell death, was observed after returning grafts to storage. Grafts stored as free scrolls retained more viable ECs than grafts prepared with the fluid bubble method (74.2± 3% vs 60.3±6%, p=0.04 (n=8). Conclusion Free scroll storage is superior to liquid bubble and partial peeling techniques. Free scrolls only showed overgrowth of ECs after 4 days in organ culture, indicating a viable time window for the clinical use of pre-prepared DMEK donor material using this method. Methods for tissue preparation and storage media developed for whole corneas should not be used in pre-prepared DMEK grafts without prior evaluation

Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers.

Clostridium difficile is a major nosocomial pathogen and the main causative agent of antibiotic-associated diarrhoea. The organism produces two potent toxins, A and B, which are its major virulence factors. These are chromosomally encoded on a region termed the pathogenicity locus (PaLoc), which also contains regulatory genes, and is absent in non-toxigenic strains. Here we show that the PaLoc can be transferred from the toxin-producing strain, 630Δerm, to three non-toxigenic strains of different ribotypes. One of the transconjugants is shown by cytotoxicity assay to produce toxin B at a similar level to the donor strain, demonstrating that a toxigenic C. difficile strain is capable of converting a non-toxigenic strain to a toxin producer by horizontal gene transfer. This has implications for the treatment of C. difficile infections, as non-toxigenic strains are being tested as treatments in clinical trials

Slim Epistemology with a Thick Skin

The distinction between ‘thick’ and ‘thin’ value concepts, and its importance to ethical theory, has been an active topic in recent meta-ethics. This paper defends three claims regarding the parallel issue about thick and thin epistemic concepts. (1) Analogy with ethics offers no straightforward way to establish a good, clear distinction between thick and thin epistemic concepts. (2) Assuming there is such a distinction, there are no semantic grounds for assigning thick epistemic concepts priority over the thin. (3) Nor does the structure of substantive epistemological theory establish that thick epistemic concepts enjoy systematic theoretical priority over the thin. In sum, a good case has yet to be made for any radical theoretical turn to thicker epistemology

Neuroinflammation and structural injury of the fetal ovine brain following intra-amniotic Candida albicans exposure.

BackgroundIntra-amniotic Candida albicans (C. Albicans) infection is associated with preterm birth and high morbidity and mortality rates. Survivors are prone to adverse neurodevelopmental outcomes. The mechanisms leading to these adverse neonatal brain outcomes remain largely unknown. To better understand the mechanisms underlying C. albicans-induced fetal brain injury, we studied immunological responses and structural changes of the fetal brain in a well-established translational ovine model of intra-amniotic C. albicans infection. In addition, we tested whether these potential adverse outcomes of the fetal brain were improved in utero by antifungal treatment with fluconazole.MethodsPregnant ewes received an intra-amniotic injection of 10(7) colony-forming units C. albicans or saline (controls) at 3 or 5 days before preterm delivery at 0.8 of gestation (term ~ 150 days). Fetal intra-amniotic/intra-peritoneal injections of fluconazole or saline (controls) were administered 2 days after C. albicans exposure. Post mortem analyses for fungal burden, peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed to determine the effects of intra-amniotic C. albicans and fluconazole treatment.ResultsIntra-amniotic exposure to C. albicans caused a severe systemic inflammatory response, illustrated by a robust increase of plasma interleukin-6 concentrations. Cerebrospinal fluid cultures were positive for C. albicans in the majority of the 3-day C. albicans-exposed animals whereas no positive cultures were present in the 5-day C. albicans-exposed and fluconazole-treated animals. Although C. albicans was not detected in the brain parenchyma, a neuroinflammatory response in the hippocampus and white matter was seen which was characterized by increased microglial and astrocyte activation. These neuroinflammatory changes were accompanied by structural white matter injury. Intra-amniotic fluconazole reduced fetal mortality but did not attenuate neuroinflammation and white matter injury.ConclusionsIntra-amniotic C. albicans exposure provoked acute systemic and neuroinflammatory responses with concomitant white matter injury. Fluconazole treatment prevented systemic inflammation without attenuating cerebral inflammation and injury

Photonic Analogue of Two-dimensional Topological Insulators and Helical One-Way Edge Transport in Bi-Anisotropic Metamaterials

Recent progress in understanding the topological properties of condensed matter has led to the discovery of time-reversal invariant topological insulators. Because of limitations imposed by nature, topologically non-trivial electronic order seems to be uncommon except in small-band-gap semiconductors with strong spin-orbit interactions. In this Article we show that artificial electromagnetic structures, known as metamaterials, provide an attractive platform for designing photonic analogues of topological insulators. We demonstrate that a judicious choice of the metamaterial parameters can create photonic phases that support a pair of helical edge states, and that these edge states enable one-way photonic transport that is robust against disorder.Comment: 13 pages, 3 figure

Governance of Ecosystem Services Across Scales in Bangladesh

Ecosystem services are governed and affected by different legal, institutional and policy frameworks. Hence, formal documented policies, regulations and statutes of Bangladesh are examined where relevant. This facilitates greater understanding of the influence that governance has on the accessibility to the benefits derived and how this might then affect livelihoods and well-being. A range of factors are found to determine effectiveness in terms of general adaptive governance principles, with coordination, enforcement and rigidity being important issues. In addition, policy development in crucial areas may not be supported by associated legal frameworks, undermining implementation. However, workable (and dynamic) combinations of primary and secondary legislation are both possible and desirable to achieve flexible policy instruments.<br/

Neuroimaging Evidence of Major Morpho-Anatomical and Functional Abnormalities in the BTBR T+TF/J Mouse Model of Autism

BTBR T+tf/J (BTBR) mice display prominent behavioural deficits analogous to the defining symptoms of autism, a feature that has prompted a widespread use of the model in preclinical autism research. Because neuro-behavioural traits are described with respect to reference populations, multiple investigators have examined and described the behaviour of BTBR mice against that exhibited by C57BL/6J (B6), a mouse line characterised by high sociability and low self-grooming. In an attempt to probe the translational relevance of this comparison for autism research, we used Magnetic Resonance Imaging (MRI) to map in both strain multiple morpho-anatomical and functional neuroimaging readouts that have been extensively used in patient populations. Diffusion tensor tractography confirmed previous reports of callosal agenesis and lack of hippocampal commissure in BTBR mice, and revealed a concomitant rostro-caudal reorganisation of major cortical white matter bundles. Intact inter-hemispheric tracts were found in the anterior commissure, ventro-medial thalamus, and in a strain-specific white matter formation located above the third ventricle. BTBR also exhibited decreased fronto-cortical, occipital and thalamic gray matter volume and widespread reductions in cortical thickness with respect to control B6 mice. Foci of increased gray matter volume and thickness were observed in the medial prefrontal and insular cortex. Mapping of resting-state brain activity using cerebral blood volume weighted fMRI revealed reduced cortico-thalamic function together with foci of increased activity in the hypothalamus and dorsal hippocampus of BTBR mice. Collectively, our results show pronounced functional and structural abnormalities in the brain of BTBR mice with respect to control B6 mice. The large and widespread white and gray matter abnormalities observed do not appear to be representative of the neuroanatomical alterations typically observed in autistic patients. The presence of reduced fronto-cortical metabolism is of potential translational relevance, as this feature recapitulates previously-reported clinical observations

The SNAPSHOT study protocol : SNAcking, Physical activity, Self-regulation, and Heart rate Over Time

Peer reviewedPublisher PD

DNA topoisomerases participate in fragility of the oncogene RET

Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APHinduced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication