A schistosome [beta] subunit remodels inactivation of a calcium channel _via_ an N-terminal polyacidic motif

The beta subunit of high voltage-activated Ca (Cav) channels targets the pore forming [alpha]~1~ subunit to the plasma membrane and defines the biophysical phenotype of the Cav channel complex. Cav channel inactivation following activation and opening is tightly regulated and is an essential property that not only prevents excessive entry of Ca^2+^ into the cell but may also have functions in signal transduction. The [beta] subunit modulates Ca^2+^-dependent and voltage-dependent components of Cav channel inactivation via its interaction with the I-II linker of the [alpha]~1~ subunit. Here, using Cav2.3 and whole-cell patch-clamp, we show that a [beta] subunit from the human parasite _Schistosoma mansoni_ ([beta]~Sm~) accelerates inactivation via a unique, long N-terminal polyacidic motif (NPAM). The accelerating effect of NPAM-containing subunits, both native ([beta]~Sm~)and chimeric mammalian [beta]~1b~, [beta]~2a~ and [beta]~3~ subunits to which NPAM had been attached, was only apparent when Ca^2+^ was internally buffered with BAPTA (5 mM) or when Ba^2+^ was used as the charge carrier, two commonly used strategies to eliminate Ca^2+^/calmodulin dependent inactivation. These results indicate that calmodulin is not involved. In addition to accelerating inactivation, NPAM-containing [beta] subunits significantly reduced current density with respect to their non NPAM-bearing counterparts. Interestingly, when the amino acids N terminal to NPAM were deleted, inactivation of Cav2.3 currents was faster than in the presence of the entire N-terminal portion of the [beta]~Sm~ subunit, as if the pre-NPAM region counteracts the effect of NPAM. Presence of NPAM also resulted in currents that activated faster, suggesting that NPAM increases open channel probability. However, NPAM does not modulate inactivation gating. In summary, this study identifies a structural determinant of Cav channel inactivation that is entirely unlike those previously known

Microscale Fluid Behavior during Cryo-EM Sample Blotting

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy for over three decades. This technique removes the excess solution from a transmission electron microscope grid by pressing absorbent filter paper against the specimen before vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

© 2008 Salvador-Recatalà et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Physiology 8 (2008): 6, doi:10.1186/1472-6793-8-6.The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory β subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the α1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two β subunit subtypes: a structurally conventional β subunit and a variant β subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavβ subunit. Here, we focus on the modulatory phenotype of the conventional Cavβ subunit (SmCavβ) using the human Cav2.3 channel as the substrate for SmCavβ and the whole-cell patch-clamp technique. The conventional Schistosoma mansoni Cavβ subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavβ run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavβ lends the Cav2.3/SmCavβ complex sensitivity to Na+ ions. A mutant version of the Cavβ subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavβ subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.This work was supported by NIH grant #s R01 AI-40522 and R01 AI-73660 to RMG and by NIH-NCRR grant # P41 RR001395 to the Biocurrents Research Center (BRC) at MBL

Observation of crystallization slowdown in supercooled para-hydrogen and ortho-deuterium quantum liquid mixtures

We report a quantitative experimental study of the crystallization kinetics of supercooled quantum liquid mixtures of para-hydrogen (pH$_2$) and ortho-deuterium (oD$_2$) by high spatial resolution Raman spectroscopy of liquid microjets. We show that in a wide range of compositions the crystallization rate of the isotopic mixtures is significantly reduced with respect to that of the pure substances. To clarify this behavior we have performed path-integral simulations of the non-equilibrium pH$_2$-oD$_2$ liquid mixtures, revealing that differences in quantum delocalization between the two isotopic species translate into different effective particle sizes. Our results provide first experimental evidence for crystallization slowdown of quantum origin, offering a benchmark for theoretical studies of quantum behavior in supercooled liquids.Comment: 6 pages, 3 figure

Sobre el impacto de la enfermedad cancerosa en la familia.

La familia ha sido identificada en la literatura como la fuente primaria de soporte psicosocial del paciente canceroso, siendo además quien asume la mayor responsabilidad en el tratamiento domiciliario. Los familiares del paciente oncológico no son observadores pasivos, sino participantes activos que pueden continuar jugando un papel vital en el tratamiento y en la recuperación del paciente

Sobre el impacto de la enfermedad cancerosa en la familia.

La familia ha sido identificada en la literatura como la fuente primaria de soporte psicosocial del paciente canceroso, siendo además quien asume la mayor responsabilidad en el tratamiento domiciliario. Los familiares del paciente oncológico no son observadores pasivos, sino participantes activos que pueden continuar jugando un papel vital en el tratamiento y en la recuperación del paciente

Near-Term Effects of Perennial Grasses on Soil Carbon and Nitrogen in Eastern Nebraska

Incorporating native perennial grasses adjacent to annual row crop systems managed on marginal lands can increase system resiliency by diversifying food and energy production. This study evaluated (1) soil organic C (SOC) and total N stocks (TN) under warm-season grass (WSG) monocultures and a low diversity mixture compared to an adjacent no-till continuous-corn system, and (2) WSG total above-ground biomass (AGB) in response to two levels of N fertilization from 2012 to 2017 in eastern Nebraska, USA. The WSG treatments consisted of (1) switchgrass (SWG), (2) big bluestem (BGB), and (3) low-diversity grass mixture (LDM; big bluestem, Indiangrass, and sideoat grama). Soils were sampled at fixed depth increments (0–120 cm) in the WSG plots and in the adjacent corn experiment in 2012 and 2017. Soil stocks (Mg ha-1) of SOC and TN were calculated on an equivalent soil mass (ESM) basis and compared within the three WSG treatments as well as between experiments (corn compared to the mean of all WSGs). Soil organic C and TN stocks within soil layers and cumulative stocks responded to the main effect of WSG (PWSG \u3c 0.05) but were no different when comparing the WSGs to corn (Pexpt = NS). Both SOC/TN stocks and cumulative stocks were generally greater in the LDM compared to the BGB. Neither SOC nor TN changed over time under either the WSGs or corn. Warm-season grass AGB responded to a three-way interaction of year, N rate, and WSG (p = 0.0007). Decreases in AGB over time were significant across WSGs and N levels except for SWG at 56 kg N ha-1 and LDM at 112 kg N ha-1. Above-ground biomass was generally greater in the LDM after the first harvest year (2013). Results suggest that incorporating WSGs into marginal cropland can maintain SOC and TN stocks while providing a significant source of biomass to be used in energy production or in integrated livestock systems

Microscale Fluid Behavior during Cryo-EM Sample Blotting

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy for over three decades. This technique removes the excess solution from a transmission electron microscope grid by pressing absorbent filter paper against the specimen before vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses

Individualized decision aid for diverse women with lupus nephritis (IDEA-WON): A randomized controlled trial.

BackgroundTreatment decision-making regarding immunosuppressive therapy is challenging for individuals with lupus. We assessed the effectiveness of a decision aid for immunosuppressive therapy in lupus nephritis.Methods and findingsIn a United States multicenter, open-label, randomized controlled trial (RCT), adult women with lupus nephritis, mostly from racial/ethnic minority backgrounds with low socioeconomic status (SES), seen in in- or outpatient settings, were randomized to an individualized, culturally tailored, computerized decision aid versus American College of Rheumatology (ACR) lupus pamphlet (1:1 ratio), using computer-generated randomization. We hypothesized that the co-primary outcomes of decisional conflict and informed choice regarding immunosuppressive medications would improve more in the decision aid group. Of 301 randomized women, 298 were analyzed; 47% were African-American, 26% Hispanic, and 15% white. Mean age (standard deviation [SD]) was 37 (12) years, 57% had annual income of <$40,000, and 36% had a high school education or less. Compared with the provision of the ACR lupus pamphlet (n = 147), participants randomized to the decision aid (n = 151) had (1) a clinically meaningful and statistically significant reduction in decisional conflict, 21.8 (standard error [SE], 2.5) versus 12.7 (SE, 2.0; p = 0.005) and (2) no difference in informed choice in the main analysis, 41% versus 31% (p = 0.08), but clinically meaningful and statistically significant difference in sensitivity analysis (net values for immunosuppressives positive [in favor] versus negative [against]), 50% versus 35% (p = 0.006). Unresolved decisional conflict was lower in the decision aid versus pamphlet groups, 22% versus 44% (p < 0.001). Significantly more patients in the decision aid versus pamphlet group rated information to be excellent for understanding lupus nephritis (49% versus 33%), risk factors (43% versus 27%), medication options (50% versus 33%; p ≤ 0.003 for all); and the ease of use of materials was higher in the decision aid versus pamphlet groups (51% versus 38%; p = 0.006). Key study limitations were the exclusion of men, short follow-up, and the lack of clinical outcomes, including medication adherence.ConclusionsAn individualized decision aid was more effective than usual care in reducing decisional conflict for choice of immunosuppressive medications in women with lupus nephritis.Trial registrationClinicaltrials.gov, NCT02319525