Identification and functional characterization of an N-terminal oligomerization domain for polycystin-2*

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C- and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function

The cAMP Signaling pathway and direct protein kinase a phosphorylation regulate polycystin-2 (TRPP2) Channel Function

Polycystin-2 (PC2) is aTRP-type, Ca2+-permeable non-selective cation channel that plays an important role in Ca2+ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus.Fil: Cantero, Maria del Rocio. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Biofísica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velázquez, Irina Florencia. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Biofísica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Streets, Andrew J.. University of Sheffield Medical School; Reino UnidoFil: Ong, Albert C. M.. University of Sheffield Medical School; Reino UnidoFil: Cantiello, Horacio Fabio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Biofísica; Argentin

Multi-scale modeling study of the source contributions to near-surface ozone and sulfur oxides levels over California during the ARCTAS-CARB period

Chronic high surface ozone (O3) levels and the increasing sulfur oxides (SOx = SO2+SO4) ambient concentrations over South Coast (SC) and other areas of California (CA) are affected by both local emissions and long-range transport. In this paper, multi-scale tracer, full-chemistry and adjoint simulations using the STEM atmospheric chemistry model are conducted to assess the contribution of local emission sourcesto SC O3 and to evaluate the impacts of transported sulfur and local emissions on the SC sulfur budgetduring the ARCTAS-CARB experiment period in 2008. Sensitivity simulations quantify contributions of biogenic and fire emissions to SC O3 levels. California biogenic and fire emissions contribute 3–4 ppb to near-surface O3 over SC, with larger contributions to other regions in CA. During a long-range transport event from Asia starting from 22 June, high SOx levels (up to ~0.7 ppb of SO2 and ~1.3 ppb of SO4) is observed above ~6 km, but they did not affect CA surface air quality. The elevated SOx observed at 1–4 km is estimated to enhance surface SOx over SC by ~0.25 ppb (upper limit) on ~24 June. The near-surface SOx levels over SC during the flight week are attributed mostly to local emissions. Two anthropogenic SOx emission inventories (EIs) from the California Air Resources Board (CARB) and the US Environmental Protection Agency (EPA) are compared and applied in 60 km and 12 km chemical transport simulations, and the results are compared withobservations. The CARB EI shows improvements over the National Emission Inventory (NEI) by EPA, but generally underestimates surface SC SOx by about a factor of two. Adjoint sensitivity analysis indicated that SO2 levels at 00:00 UTC (17:00 local time) at six SC surface sites were influenced by previous day maritime emissions over the ocean, the terrestrial emissions over nearby urban areas, and by transported SO2 from the north through both terrestrial and maritime areas. Overall maritime emissions contribute 10–70% of SO2 and 20–60% fine SO4 on-shore and over the most terrestrial areas, with contributions decreasing with in-land distance from the coast. Maritime emissions also modify the photochemical environment, shifting O3 production over coastal SC to more VOC-limited conditions. These suggest an important role for shipping emission controls in reducing fine particle and O3concentrations in SC

Transboundary health impacts of transported global air pollution and international trade

Millions of people die every year from diseases caused by exposure to outdoor air pollution1, 2, 3, 4, 5. Some studies have estimated premature mortality related to local sources of air pollution6, 7, but local air quality can also be affected by atmospheric transport of pollution from distant sources8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. International trade is contributing to the globalization of emission and pollution as a result of the production of goods (and their associated emissions) in one region for consumption in another region14, 19, 20, 21, 22. The effects of international trade on air pollutant emissions23, air quality14 and health24 have been investigated regionally, but a combined, global assessment of the health impacts related to international trade and the transport of atmospheric air pollution is lacking. Here we combine four global models to estimate premature mortality caused by fine particulate matter (PM2.5) pollution as a result of atmospheric transport and the production and consumption of goods and services in different world regions. We find that, of the 3.45 million premature deaths related to PM2.5 pollution in 2007 worldwide, about 12 per cent (411,100 deaths) were related to air pollutants emitted in a region of the world other than that in which the death occurred, and about 22 per cent (762,400 deaths) were associated with goods and services produced in one region for consumption in another. For example, PM2.5 pollution produced in China in 2007 is linked to more than 64,800 premature deaths in regions other than China, including more than 3,100 premature deaths in western Europe and the USA; on the other hand, consumption in western Europe and the USA is linked to more than 108,600 premature deaths in China. Our results reveal that the transboundary health impacts of PM2.5 pollution associated with international trade are greater than those associated with long-distance atmospheric pollutant transport

The QuinteT Recruitment Intervention supported five randomized trials to recruit to target: a mixed-methods evaluation

ObjectiveTo evaluate the impact of the Quintet Recruitment Intervention (QRI) on recruitment in challenging randomized controlled trials (RCTs) that have applied the intervention. The QRI aims to understand recruitment difficulties, and then implements ‘QRI-actions’ to address these as recruitment proceeds.Study Design and SettingA mixed-methods study, comprising: a) before-and-after comparisons of recruitment rates and numbers of patients approached, and b) qualitative case studies, including documentary analysis and interviews with RCT investigators.ResultsFive UK-based publicly-funded RCTs were included in the evaluation. All recruited to target. RCT2 and RCT5 both received up-front pre-recruitment training before the intervention was applied. RCT2 did not encounter recruitment issues and recruited above target from its outset. Recruitment difficulties, particularly communication issues, were identified and addressed through QRI-actions in RCTs 1, 3, 4 and 5. Randomization rates significantly improved post-QRI-action in RCTs 1,3, and 4. QRI-actions addressed issues with approaching eligible patients in RCTs 3 and 5, which both saw significant increases in patients approached. Trial investigators reported that the QRI had unearthed issues they had been unaware of, and reportedly changed their practices post QRI-action.ConclusionThere is promising evidence to suggest the QRI can support recruitment to difficult RCTs. This needs to be substantiated with future controlled evaluations

Thiazolidinediones inhibit MDCK cyst growth through disrupting oriented cell division and apicobasal polarity

Thiazolidinediones have been reported to retard cystic disease in rodent models by uncertain mechanisms. We hypothesized that their major effect in retarding cystogenesis was through inhibiting cell proliferation or stimulating apoptosis. In the Madin-Darby canine kidney cell (MDCK) model, rosiglitazone inhibited cyst growth in a time- and dose-dependent manner and this was accompanied by a reduction in basal proliferation and an increase in apoptosis. Unexpectedly, we also observed a striking abnormality in lumen formation resulting in a characteristic multiple lumen or loss of lumen phenotype in treated cells at doses which did not inhibit cell proliferation. These changes were preceded by mislocalization of gp135 and Cdc42, misorientation of the mitotic spindle, and retardation in centrosome reorientation with later changes in primary cilia length and mislocalization of E-cadherin. Cdc42 activation was unaffected by rosiglitazone in monolayer culture but was profoundly inhibited in three-dimensional culture. MDCK cells stably expressing mutant Cdc42 showed a similar mislocalization of gp135 expression and multilumen phenotype in the absence of rosiglitazone. We conclude that rosiglitazone influences MDCK cyst growth by multiple mechanisms involving dosage-dependent effects on proliferation, spindle orientation, centrosome migration, and lumen formation. Correct spatial Cdc42 activation is critical for lumen formation, but the effect of rosiglitazone is likely to involve both Cdc42 and non-Cdc42 pathways