Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na(+)) and, indirectly, serum potassium (K(+)) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ((22)Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive (22)Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K(+), the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. (22)Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion

Long-term regulation of proximal tubule acid–base transporter abundance by angiotensin II

In the proximal tubule, angiotensin II (Ang-II) regulates HCO−3 reabsorption and H+ secretion by binding the type 1 Ang-II (AT1) receptor, stimulating Na+/HCO−3 cotransport and Na+/H+ exchange. Studies were carried out to determine if long-term changes in Ang-II receptor occupation alter the abundance of the basolateral Na+/HCO−3 cotransporter (NBC1) or the apical membrane type 3Na+/H+ exchanger (NHE3). In the first set of experiments, rats eating a low-sodium diet were infused with the AT1 blocker, candesartan, or vehicle. In the second, lisinopril-infused rats were infused with either Ang II or vehicle. Transporter abundances were determined in whole kidney homogenates (WKH) and in brush border membrane (BBM) preparations by semiquantitative immunoblotting. Tissue distribution of transporters was assessed by immunocytochemistry. Blockade of the AT1 receptor by candesartan caused decreased abundance of NBC1 in WKH (59±9% of control; P<0.05) and Ang-II infusion increased abundance (130±7% of control; P<0.05). Changes in NBC1 in response to candesartan were confirmed immunohistochemically. Neither candesartan nor Ang II infusion affected the abundance of NHE3 in WKH or cortical homogenates. Candesartan decreased type 2 sodium-phosphate cotransporter abundance in both WKH (52±7% of control; P<0.05) and BBM (32±7% of control; P<0.05). Serum bicarbonate was decreased by candesartan and increased by Ang-II. Candesartan also decreased urinary ammonium excretion (P<0.05). The long-term effects of Ang-II in the proximal tubule may be mediated in part by regulation of NBC1 abundance, modifying bicarbonate reabsorption

The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation.

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na(+) and K(+) homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function

Confinement of matroid representations to subsets of partial fields

Let M be a matroid representable over a (partial) field P and B a matrix representable over a sub-partial field P' of P. We say that B confines M to P' if, whenever a P-representation matrix A of M has a submatrix B, A is a scaled P'-matrix. We show that, under some conditions on the partial fields, on M, and on B, verifying whether B confines M to P' amounts to a finite check. A corollary of this result is Whittle's Stabilizer Theorem. A combination of the Confinement Theorem and the Lift Theorem from arXiv:0804.3263 leads to a short proof of Whittle's characterization of the matroids representable over GF(3) and other fields. We also use a combination of the Confinement Theorem and the Lift Theorem to prove a characterization, in terms of representability over partial fields, of the 3-connected matroids that have k inequivalent representations over GF(5), for k = 1, ..., 6. Additionally we give, for a fixed matroid M, an algebraic construction of a partial field P_M and a representation A over P_M such that every representation of M over a partial field P is equal to f(A) for some homomorphism f:P_M->P. Using the Confinement Theorem we prove an algebraic analog of the theory of free expansions by Geelen et al.Comment: 45 page

A complex systems model of breast cancer etiology: The Paradigm II Model

BACKGROUND: Complex systems models of breast cancer have previously focused on prediction of prognosis and clinical events for individual women. There is a need for understanding breast cancer at the population level for public health decision-making, for identifying gaps in epidemiologic knowledge and for the education of the public as to the complexity of this most common of cancers. METHODS AND FINDINGS: We developed an agent-based model of breast cancer for the women of the state of California using data from the U.S. Census, the California Health Interview Survey, the California Cancer Registry, the National Health and Nutrition Examination Survey and the literature. The model was implemented in the Julia programming language and R computing environment. The Paradigm II model development followed a transdisciplinary process with expertise from multiple relevant disciplinary experts from genetics to epidemiology and sociology with the goal of exploring both upstream determinants at the population level and pathophysiologic etiologic factors at the biologic level. The resulting model reproduces in a reasonable manner the overall age-specific incidence curve for the years 2008-2012 and incidence and relative risks due to specific risk factors such as BRCA1, polygenic risk, alcohol consumption, hormone therapy, breastfeeding, oral contraceptive use and scenarios for environmental toxin exposures. CONCLUSIONS: The Paradigm II model illustrates the role of multiple etiologic factors in breast cancer from domains of biology, behavior and the environment. The value of the model is in providing a virtual laboratory to evaluate a wide range of potential interventions into the social, environmental and behavioral determinants of breast cancer at the population level. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose

Field Measurements of Terrestrial and Martian Dust Devils

Surface-based measurements of terrestrial and martian dust devils/convective vortices provided from mobile and stationary platforms are discussed. Imaging of terrestrial dust devils has quantified their rotational and vertical wind speeds, translation speeds, dimensions, dust load, and frequency of occurrence. Imaging of martian dust devils has provided translation speeds and constraints on dimensions, but only limited constraints on vertical motion within a vortex. The longer mission durations on Mars afforded by long operating robotic landers and rovers have provided statistical quantification of vortex occurrence (time-of-sol, and recently seasonal) that has until recently not been a primary outcome of more temporally limited terrestrial dust devil measurement campaigns. Terrestrial measurement campaigns have included a more extensive range of measured vortex parameters (pressure, wind, morphology, etc.) than have martian opportunities, with electric field and direct measure of dust abundance not yet obtained on Mars. No martian robotic mission has yet provided contemporaneous high frequency wind and pressure measurements. Comparison of measured terrestrial and martian dust devil characteristics suggests that martian dust devils are larger and possess faster maximum rotational wind speeds, that the absolute magnitude of the pressure deficit within a terrestrial dust devil is an order of magnitude greater than a martian dust devil, and that the time-of-day variation in vortex frequency is similar. Recent terrestrial investigations have demonstrated the presence of diagnostic dust devil signals within seismic and infrasound measurements; an upcoming Mars robotic mission will obtain similar measurement types

Archaeological signatures of landscape and settlement change on the Isle of Harris

Between 2004 and 2011, a programme of archaeological investigation by the University of Birmingham on the Isle of Harris, a distinctive island forming part of the Western Isles of Scotland, has allowed the archaeological remains of this enigmatic place to be further characterised and understood. Despite intensive archaeological interest in the archipelago for a number of decades, the Isle of Harris has been overlooked and only now are we beginning to identify the archaeological resource and make comparisons to the wealth of published data from islands such as the Uists, Barra and Lewis. This paper highlights some generic overall patterns of archaeological signatures on the Isle which has been identified through a range of archaeological methods including field walking, intrusive excavation, aerial reconnaissance, geophysical and topographical survey, and documentary research. Several key case studies will be introduced including upland shieling complexes and mulitperiod settlement sites on the west coast machair systems. The purpose of the paper is not to present a gazetteer of the results of the work to date, but to highlight some of the key findings with a view to demonstrating that the Isle of Harris is directly comparable with the archaeologically rich landscapes of the other islands

Temporal evolution of solar energetic particle spectra

During solar flares and coronal mass ejections, solar energetic par- ticles (SEPs) may be released into the interplanetary medium and near-Earth locations. The energy spectra of SEP events at 1 AU are typically averaged over the entire event or studied in a few snapshots. In this paper we analyze the time evolution of the energy spectra of four large selected SEP events using a large number of snapshots. We use a multi-spacecraft and multi-instrument approach for the observations, obtained over a wide SEP energy range. We find large differences in the spectra at the beginning of the events as measured by different instruments. We show that over time, a wave-like structure is observed traveling through the spectra from the highest energies to the lowest energies, creating an “arch” shape which then straightens into a power law later in the event, after times of the order of 10 hours. We discuss the processes that determine SEP intensities and their role in shaping the spectral time evolution

Analytically Solvable Asymptotic Model of Atrial Excitability

We report a three-variable simplified model of excitation fronts in human atrial tissue. The model is derived by novel asymptotic techniques \new{from the biophysically realistic model of Courtemanche et al (1998) in extension of our previous similar models. An iterative analytical solution of the model is presented which is in excellent quantitative agreement with the realistic model. It opens new possibilities for analytical studies as well as for efficient numerical simulation of this and other cardiac models of similar structure