Compromised Phagosome Maturation Underlies RPE Pathology in Cell Culture and Whole Animal Models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group

Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: apical secretion and distribution

Pigment epithelium-derived factor (PEDF) is an extracellular protein derived from the retinal pigment epithelium (RPE), a tissue formed by polarized cells that release growth and trophic factors in a directional fashion. We have investigated the distribution and directional release of PEDF protein by the monkey RPE. We established primary cultures of monkey RPE cells that expressed the PEDF gene, and that synthesized and secreted the PEDF protein. Northern analysis of RPE cultures and monkey ocular tissues showed that PEDF transcripts were highly expressed in RPE as compared with several other monkey ocular tissues, being even more abundant in cultured cells than they were in the native RPE. The differentiated RPE cells in culture secreted protein that shared the immunological, biochemical and biological characteristics of PEDF. The overall PEDF levels in the RPE conditioned media reached 6.5 mg ml- after 8 days in culture (i.e. 1.1 pg of PEDF per RPE cell). RPE cells were cultivated on permeable supports as monolayers forming a barrier between apical and basal compartments. Apical and basal culture media were sampled at three or four-day intervals for 18 cycles, and the PEDF content was quantified. Most of the PEDF protein was significantly higher in the apical than in the basal medium (>4 times) at the initial recovery intervals, to be detected only in the apical medium at the latter intervals. In the native monkey eye, the concentration of soluble PEDF in the interphotoreceptor matrix (144 nM) was 7-fold and 25-fold greater than in vitreous and aqueous, respectively. PEDF was abundant in the interphotoreceptor matrix surrounding rod and cone outer segments, and was detectable at lower levels in the RPE as visualized by confocal microscopy. We concluded that PEDF synthesized by the RPE is secreted preferentially from the apical surface and is distributed apically to the RPE bordering the outer segments of photoreceptors. PEDF can be a useful marker for RPE polarization and differentiation. The polarization of RPE may be an important mechanism to control PEDF secretion and our results offer interesting possibilities on regulation of PEDF

Robust Lysosomal Calcium Signaling Through Channel TRPML1 is Impaired by Lysosomal Lipid Accumulation

The transient receptor potential cation channelmucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calciuminRPE cells andwhether such release is affectedby lysosomal accumulations.The TRPM LagonistML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-L-phenylalanine-b-naphthylamide. Treatment with desipramine toinhibit acidsphingomyelinase orYM201636 to inhibitPIKfyve also reducedthe cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML-/- mice showed no response to ML-SA1. Cotreatmentwith chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomalCa2+ release.LysosomalCa2+ releasewas also impaired in RPEcells from the ATP-binding cassette, subfamily A, member 4-/-mouse model of Stargardt\u27s retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models,suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calciumlevels in RPE cells, but this response is reduced by lysosomal accumulation.-Gomez, N. M.,Lu,W.Lim, J. C.,Kiselyov, K.,Campagno, K.E.,Grishchuk,Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation. FASEB J. 32, 782-794 (2018). www.fasebj.org. © FASEB

Characteristics of the National Applicant Pool for Clinical Informatics Fellowships (2016-2017)

We conducted a national study to assess the numbers and diversity of applicants for 2016 and 2017 clinical informatics fellowship positions. In each year, we collected data on the number of applications that programs received from candidates who were ultimately successful vs. unsuccessful. In 2017, we also conducted an anonymous applicant survey. Successful candidates applied to an average of 4.2 and 5.5 programs for 2016 and 2017, respectively. In the survey, unsuccessful candidates reported applying to fewer programs. Assuming unsuccessful candidates submitted between 2-5 applications each, the total applicant pool numbered 42-69 for 2016 (competing for 24 positions) and 52-85 for 2017 (competing for 30 positions). Among survey respondents (n=33), 24% were female, 1 was black and none were Hispanic. We conclude that greater efforts are needed to enhance interest in clinical informatics among medical students and residents, particularly among women and members of underrepresented minority groups

Real-life biomarker response to anti-IL5 and anti-IgE therapies in severe asthma patients

Funding: This study was conducted by the Observational and Pragmatic Research Institute (OPRI) Pte Ltd and was partially funded by Optimum Patient Care Global and AstraZeneca Ltd. No funding was received by OPRI for its contribution.Peer reviewedPostprin

The theory of the firm and its critics: a stocktaking and assessment

Includes bibliographical references."Prepared for Jean-Michel Glachant and Eric Brousseau, eds. New Institutional Economics: A Textbook, Cambridge, Cambridge University Press.""This version: August 22, 2005."Since its emergence in the 1970s the modern economic or Coasian theory of the firm has been discussed and challenged by sociologists, heterodox economists, management scholars, and other critics. This chapter reviews and assesses these critiques, focusing on behavioral issues (bounded rationality and motivation), process (including path dependence and the selection argument), entrepreneurship, and the challenge from knowledge-based theories of the firm

Biomarker-defined clusters by level of Type 2 inflammatory involvement in severe asthma

Peer reviewedPostprin

Pre-Micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma–associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS

Gendering the careers of young professionals: some early findings from a longitudinal study. in Organizing/theorizing: developments in organization theory and practice

Wonders whether companies actually have employees best interests at heart across physical, mental and spiritual spheres. Posits that most organizations ignore their workforce – not even, in many cases, describing workers as assets! Describes many studies to back up this claim in theis work based on the 2002 Employment Research Unit Annual Conference, in Cardiff, Wales