LEDGF1-326 Decreases P23H and Wild Type Rhodopsin Aggregates and P23H Rhodopsin Mediated Cell Damage in Human Retinal Pigment Epithelial Cells

P23H rhodopsin, a mutant rhodopsin, is known to aggregate and cause retinal degeneration. However, its effects on retinal pigment epithelial (RPE) cells are unknown. The purpose of this study was to determine the effect of P23H rhodopsin in RPE cells and further assess whether LEDGF(1-326), a protein devoid of heat shock elements of LEDGF, a cell survival factor, reduces P23H rhodopsin aggregates and any associated cellular damage.ARPE-19 cells were transiently transfected/cotransfected with pLEDGF(1-326) and/or pWT-Rho (wild type)/pP23H-Rho. Rhodopsin mediated cellular damage and rescue by LEDGF(1-326) was assessed using cell viability, cell proliferation, and confocal microscopy assays. Rhodopsin monomers, oligomers, and their reduction in the presence of LEDGF(1-326) were quantified by western blot analysis. P23H rhodopsin mRNA levels in the presence and absence of LEDGF(1-326) was determined by real time quantitative PCR.P23H rhodopsin reduced RPE cell viability and cell proliferation in a dose dependent manner, and disrupted the nuclear material. LEDGF(1-326) did not alter P23H rhodopsin mRNA levels, reduced its oligomers, and significantly increased RPE cell viability as well as proliferation, while reducing nuclear damage. WT rhodopsin formed oligomers, although to a smaller extent than P23H rhodopsin. Further, LEDGF(1-326) decreased WT rhodopsin aggregates.P23H rhodopsin as well as WT rhodopsin form aggregates in RPE cells and LEDGF(1-326) decreases these aggregates. Further, LEDGF(1-326) reduces the RPE cell damage caused by P23H rhodopsin. LEDGF(1-326) might be useful in treating cellular damage associated with protein aggregation diseases such as retinitis pigmentosa

Comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system

<p>Abstract</p> <p>Background</p> <p>Bacteria used as indicators for pathogenic microorganisms in water are not considered adequate as enteric virus indicators. Surface water from a tropical high-altitude system located in Mexico City that receives rainwater, treated and non-treated wastewater used for irrigation, and groundwater used for drinking, was studied.</p> <p>Methods</p> <p>The presence of enterovirus, rotavirus, astrovirus, coliphage, coliform bacteria, and enterococci was determined during annual cycles in 2001 and 2002. Enteric viruses in concentrated water samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Coliphages were detected using the double agar layer method. Bacteria analyses of the water samples were carried out by membrane filtration.</p> <p>Results</p> <p>The presence of viruses and bacteria in the water used for irrigation showed no relationship between current bacterial indicator detection and viral presence. Coliphages showed strong association with indicator bacteria and enterovirus, but weak association with other enteric viruses. Enterovirus and rotavirus showed significant seasonal differences in water used for irrigation, although this was not clear for astrovirus.</p> <p>Conclusion</p> <p>Coliphages proved to be adequate faecal pollution indicators for the irrigation water studied. Viral presence in this tropical high-altitude system showed a similar trend to data previously reported for temperate zones.</p

Multivariable regression analysis in Schistosoma mansoni-infected individuals in the Sudan reveals unique immunoepidemiological profiles in uninfected, egg+ and non-egg+ infected individuals

Background: In the Sudan, Schistosoma mansoni infections are a major cause of morbidity in schoolaged children and infection rates are associated with available clean water sources. During infection, immune responses pass through a Th1 followed by Th2 and Treg phases and patterns can relate to different stages of infection or immunity. Methodology: This retrospective study evaluated immunoepidemiological aspects in 234 individuals(range 4–85 years old) from Kassala and Khartoum states in 2011. Systemic immune profiles(cytokines and immunoglobulins) and epidemiological parameters were surveyed in n = 110 persons presenting patent S. mansoni infections (egg+), n = 63 individuals positive for S. mansoni via PCR in sera but egg negative (SmPCR+) and n = 61 people who were infection-free (Sm uninf). Immunoepidemiological findings were further investigated using two binary multivariable regression analysis. Principal Findings: Nearly all egg+ individuals had no access to latrines and over 90% obtained water via the canal stemming from the Atbara River. With regards to age, infection and an egg+ status was linked to young and adolescent groups. In terms of immunology, S. mansoni infection per se was strongly associated with increased SEA-specific IgG4 but not IgE levels. IL-6, IL-13 and IL-10 were significantly elevated in patently-infected individuals and positively correlated with egg load. In contrast, IL-2 and IL-1β were significantly lower in SmPCR+ individuals when compared to Sm uninf and egg+ groups which was further confirmed during multivariate regression analysis. Conclusions/Significance: Schistosomiasis remains an important public health problem in the Sudan with a high number of patent individuals. In addition, SmPCR diagnostics revealed another cohort of infected individuals with a unique immunological profile and provides an avenue for future studies on non-patent infection states. Future studies should investigate the downstream signalling pathways/mechanisms of IL-2 and IL-1β as potential diagnostic markers in order to distinguish patent from non-patent individuals

PI3K and ERK-Induced Rac1 Activation Mediates Hypoxia-Induced HIF-1α Expression in MCF-7 Breast Cancer Cells

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1

Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs

Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (RhP23H). Unlike the wild-type Rh, mutant RhP23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded RhP23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that RhP23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1P37H (the equivalent of mammalian RhP23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1P37H retina. Despite this, Rh1P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1P37H retina. Pharmacological treatment of Rh1P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration