Frontiers in Physiology

Published by Frontiers
Online ISSN: 1664-042X
Phenotypic expression of stress effects on the panicle of sensitive HHB67 (= 843A × H77/833-2) and an improved version of this hybrid (843A × ICMR 01029), and showing a better seed set and a better grain filling in the tolerant hybrid. ICMR01029 is an introgression line with a terminal drought tolerance on linkage group 2 from donor parent PRLT/89-33, after four backcrosses using H77/833-2 (Source: Hash, unpublished).
Strategy for the development of a skeleton map and identification of drought tolerance QTLs (Source: Hash, unpublished).
The outcome of QTL mapping: identification of a set of QTLs for different traits (leaf rolling, biomass yield, dry straw yield, panicle number, panicle grain number, 100-grain mass, grain yield, and harvest index) in pearl millet in three drought nursery experiments.
Scheme for marker-assisted backcrossing of desired (+) segregants. A QTL was introgressed after four rounds of marker-assisted backcrossing (Source: Hash, unpublished).
Typical line source experiment, which allows the imposition of a gradient of watering regimes, from fully irrigated conditions close to the irrigation line, to severely stressed at the point most distant from the line (Source: SMH Rizvi, unpublished).
Pearl millet is highly resilient to some of the driest areas of the world, like the Sahel area or fringes of the Thar desert in India. Despite this, there is a wealth of variation in pearl millet genotypes for their adaptation to drought, and the object of this paper was to review some related work in the past 25 years to harness these capacities towards the breeding of better adapted cultivars. Work on short duration cultivars has been a major effort. Pearl millet has also some development plasticity thanks to a high tillering ability, which allows compensating for possible drought-related failure of the main culm under intermittent drought. The development of molecular tools for breeding has made great progress in the last 10-15 years and markers, maps, EST libraries, BACs are now available and a number of QTLs for different traits, including drought, have been identified. Most of the work on drought has focused on the drought tolerance index (DTI), an index that reflect the genetic differences in drought adaptation that are independent of flowering time and yield potential. The DTI is closely associated to the panicle harvest index (PNHI), a trait that relates to a better grain setting and grain filling capacity. Initial work on the DTI involved empirical breeding and selection based on PNHI. A QTL for PNHI has then been identified and introgressed by marker-assisted backcrossing. More recently, a thorough dissection of that QTL has been carried out and shows that high PNHI is related to the constitutive ability of tolerant lines to save water (lower leaf conductance and sensitivity of transpiration to high vapor pressure deficit) at a vegetative stage and use it for the grain filling period. However, there is no contribution of root traits in this QTL. Current work is taking place to map these water saving traits, understand their genetic interactions, and design ideotypes having specific genetic make up towards adaptation to specific rainfall environments.
Pluripotency in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is regulated by three transcription factors-OCT3/4, SOX2, and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behavior of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11) and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency.
| γPKC is translocated around the centrosome in microtubuleand PKC kinase activity-dependent manners. (A) Time-lapse imaging of γPKC-GFP expressing HeLa cells treated with TPA. γPKC-GFP expressing cells were treated with 100 nM TPA for 1 h. These images were obtained from the same cells at each time point (0, 15, 30, and 60 min) after TPA treatment. The translocations of γPKC-GFP to the plasma membrane (15 min) and to the perinuclear region (30 and 60 min) were observed, accompanied by a reduction in cell size (30 and 60 min). Bar = 10 μm. (B) γPKC translocation around the centrosome. Representative fluorescence images (γPKC-GFP, γ-tubulin, and merged) of γPKC-GFP-expressing HeLa cells treated with 100 nM TPA for 60 min, followed by immunostaining with an anti-γ-tubulin antibody, a marker of the centrosome. Translocated γPKC-GFP was localized around the centrosome. Bar = 10 μm. (C-E) γPKC translocation around the centrosome and cell shrinkage were dependent on microtubules
γ PKC is translocated around the centrosome in microtubule- and PKC kinase activity-dependent manners. (A) Time-lapse imaging of γPKC-GFP expressing HeLa cells treated with TPA. γPKC-GFP expressing cells were treated with 100 nM TPA for 1 h. These images were obtained from the same cells at each time point (0, 15, 30, and 60 min) after TPA treatment. The translocations of γPKC-GFP to the plasma membrane (15 min) and to the perinuclear region (30 and 60 min) were observed, accompanied by a reduction in cell size (30 and 60 min). Bar = 10 μm. (B) γPKC translocation around the centrosome. Representative fluorescence images (γPKC-GFP, γ-tubulin, and merged) of γPKC-GFP-expressing HeLa cells treated with 100 nM TPA for 60 min, followed by immunostaining with an anti-γ-tubulin antibody, a marker of the centrosome. Translocated γPKC-GFP was localized around the centrosome. Bar = 10 μm. (C–E) γPKC translocation around the centrosome and cell shrinkage were dependent on microtubules and PKC kinase activity. (C) Representative fluorescence images of γPKC-GFP-expressing cells treated with TPA alone (left), TPA plus 1 μg/ml nocodazole, an inhibitor of tubulin polymerization (center) and TPA plus 2.5 μg/ml Gö6976, an inhibitor of cPKC (right). Nocodazole and Gö6976 were added 20 min prior to treatment with 100 nM TPA. The cells were fixed 30 min after TPA treatment. Nocodazole and Gö6976 prevented the TPA-induced perinuclear translocation of γPKC-GFP and cell shrinkage. Bar = 10 μm. (D) The percentage of γPKC-GFP-positive HeLa cells with perinuclear translocation after 30 min of TPA treatment, shown in (C). The data represent the mean ± standard error from four independent experiments. (E) The cell size of the GFP-positive HeLa cells after TPA treatment for 30 min, shown in (C). The data represent the mean ± standard error. The number of quantified cells in each treatment is indicated in the column. **p < 0.01, ***p < 0.001 (unpaired t-test).
SCA14 mutant γ PKC impairs the perinuclear translocation and cell size reduction in response to TPA treatment. (A) Representative fluorescence images of GFP (left), WT γPKC-GFP (center) or S119P γPKC-GFP (right)-expressing HeLa cells treated with TPA for 30 min. In response to TPA treatment for 30 min, WT γPKC-GFP exhibited membrane translocation, followed by perinuclear translocation and a reduction in cell size. In contrast, S119P mutant γPKC-GFP did not exhibit perinuclear translocation and remained at the plasma membrane. In cells expressing GFP alone, TPA did not induce the translocation of GFP or cell shrinkage. Scale bar = 10 μm. (B, C) Quantification of the TPA-induced perinuclear translocation of γPKC-GFP and cell shrinkage. (B) The percentage of γPKC-GFP-positive HeLa cells with perinuclear translocation, shown in (A). WT and mutant (S119F, S119P, and G128D) γPKC-GFP were expressed in HeLa cells and were stimulated with TPA for 30 min. After fixation, the percentage of cells with perinuclear translocation was evaluated. The data represent the mean ± standard error from three independent experiments. (C) The cell size of the GFP-positive HeLa cells after TPA treatment, shown in (A). GFP, WT, and mutant γPKC-GFP were expressed in HeLa cells and were stimulated with TPA for 30 min. After fixation, the cell size was measured from the GFP fluorescence images. The data represent the mean ± standard error. The number of quantified cells in each treatment is indicated in the column. **p < 0.01, ***p < 0.001 (unpaired t-test).
γ PKC, but not SCA14 mutant γ PKC, mediates the TPA-induced remodeling of the actin cytoskeleton and the activation of macropinocytosis. (A) Representative images (DIC, γPKC-GFP, F-actin, and merged) of γPKC-GFP-expressing HeLa cells treated with TPA for 30 min. The lower panels show high magnification images of the boxed areas in the merged images in the upper panels. F-actin was visualized by staining with 100 nM phalloidin-TRITC. Perinuclear γPKC-GFP was colocalized with F-actin around many large vesicles (> 0.2 μm in a diameter). The scale bars in the upper and lower panels are 10 and 2 μm, respectively. (B) FITC-dextran uptake in γPKC-HT-expressing cells in response to TPA treatment. FITC-dextran, a fluorescent marker for macropinocytosis, was simultaneously incubated with cells expressing γPKC-HT for 30 min during TPA treatment. Representative images (γPKC-HT, FITC-dextran and merged) are shown. The far right image shows the high magnification image of the boxed area in the merged image. FITC-dextran was surrounded by γPKC-HT. The scale bars in the merged and high magnification images are 5 and 2 μm, respectively. (C) FITC-dextran uptake in WT and mutant (S119P and G128D) γPKC-HT-expressing cells in response to TPA treatment. Cells expressing γPKC-HT were simultaneously incubated for 30 min with FITC-dextran and TPA. Representative images (γPKC-HT and FITC-dextran) are shown. TPA enhanced the uptake of FITC-dextran in cells expressing WT γPKC-HT, but not in cells expressing mutant γPKC-HT. Scale bar = 5 μm.
γ PKC, but not SCA14 mutant γ PKC, mediates the TPA-induced perinuclear translocation of phosphorylated MARCKS. (A) Perinuclear translocation of phosphorylated MARCKS (p-MARCKS) 30 min after TPA treatment. Representative images (γPKC-GFP, p-MARCKS and merged) are shown. Cells expressing γPKC-GFP were treated with TPA for 30 min, followed by immunostaining with an anti-p-MARCKS antibody. The far right image shows the high magnification image of the boxed area in the merged image. p-MARCKS was strongly colocalized with the perinuclear γPKC-GFP, especially around the vesicles. The scale bars of the merged and high magnification images are 5 and 2 μm, respectively. (B) Localization of p-MARCKS in cells expressing GFP alone, WT and S119P γPKC-GFP 30 min after TPA treatment. Representative images (p-MARCKS and GFP) are shown. As described above, p-MARCKS was colocalized with WT γPKC-GFP in the perinuclear region. In contrast, obvious p-MARCKS immunostaining was not observed in cells expressing GFP alone or mutant γPKC-GFP. Scale bar = 10 μm.
Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca(2+) and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate), a type of phorbol ester. Wild-type (WT) γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in pathogenesis of SCA14.
| Comparing age, parity, educational levels, prenatal care, anemia, and placenta previa between the cases and controls.
A retrospective case-control study was conducted to investigate the risk factors for pre-eclampsia - including the protective effect of placenta previa - at Medani Maternity Hospital, Sudan. Medical files of the patients during the period 2003-2010 were reviewed for age, parity, education level, prenatal care, placenta previa, and hemoglobin level. Women with pre-eclampsia were the cases, and women with normal pregnancy were the controls. There were 54,339 singleton deliveries and 1765 women with pre-eclampsia in the hospital, giving the incidence of pre-eclampsia of 3.2%. The risk factors for pre-eclampsia were; women with age >35 years (OR = 1.4, 95% CI: 1.1-1.8), primiparity (OR = 3.3, 95% CI: 2.7-4.0), para >5 (OR = 3.1, 95% CI: 2.4-4.0), and anemia (OR = 3.3, 95% CI: 2.8-3.9). The risk of pre-eclampsia was inversely increased with education level and prenatal care attendance. The prevalence of placenta previa was 0 (0%) and 55 (3.3%), < 0.001 in pre-eclamptic and control women, respectively. Placenta previa was a significant protective factor of pre-eclampsia (OR = 0.3, 95% CI: 0.1-0.7). Although, the socio-demographic risk factors for pre-eclampsia observed among women at Medani hospital were similar to those found in other settings; placenta previa was associated with decreased incidence of pre-eclampsia.
Schematic model illustrating the relationship between extranuclear and nuclear actions of E2 on target cells. E2, 17β-estradiol; mER, estrogen receptor located at the plasma membrane; ER, estrogen receptor; AP-1, activating factor-1. For details, see text.
Schematic model illustrating the ERβ-dependent E2-induced neuroprotection against oxidative stress. Exposure to oxidative stress induces cytochrome c (cyt c) release into the cytoplasm. Once in the cytosol cyt c mediates the activation of the adaptor molecule apoptosis–protease activating factor-1 (Apaf-1), generating the apoptosome. Apoptosome can recruit caspase-9 favoring proteinase activation. These events induce the catalytic maturation of caspase-3, which mediates the biochemical and morphological features of apoptosis. E2, via the synergy between extranuclear and nuclear ERβ activities, increases neuroglobin (Ngb) levels, reallocates Ngb at mitochondria, facilitates Ngb-cytochrome c interaction, and prevents apoptosome formation. For details, see text.
Besides its crucial role in many physiological events, 17β-estradiol (E2) exerts protective effects in the central nervous system. The E2 effects are not restricted to the brain areas related with the control of reproductive function, but rather are widespread throughout the developing and the adult brain. E2 actions are mediated through estrogen receptors (i.e., ERα and ERβ) belonging to the nuclear receptor super-family. As members of the ligand-regulated transcription factor family, classically, the actions of ERs in the brain were thought to mediate only the E2 long-term transcriptional effects. However, a growing body of evidence highlighted rapid, membrane initiated E2 effects in the brain that are independent of ER transcriptional activities and are involved in E2-induced neuroprotection. The aim of this review is to focus on the rapid effects of E2 in the brain highlighting the specific role of the signaling pathway(s) of the ERβ subtype in the neuroprotective actions of E2.
Two different isoforms of the estrogen receptors (i.e., ERα and ERβ) mediate pleiotropic 17β-estradiol (E2)-induced cellular effects. The ERs are principally localized in the nucleus where they act by globally modifying the expression of the E2-target genes. The premise that E2 effects are exclusively mediated through the nuclear localized ERs has been rendered obsolete by research over the last 15 years demonstrating that ERα and ERβ proteins are also localized at the plasma membranes and in other extra-nuclear organelles. The E2 modulation of cancer cell proliferation represents a good example of the impact of membrane-initiated signals on E2 effects. In fact, E2 via ERα elicits rapid signals driving cancer cells to proliferation (e.g., in breast cancer cells), while E2-induced ERβ rapid signaling inhibits proliferation (e.g., in colon cancer cells). In this review we provide with an overview of the complex system of E2-induced signal transduction pathways, their impact on E2-induced cancer cell proliferation, and the participation of E2-induced membrane-initiated signals in tumor environment.
The successful establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. In order to understand this process we have studied the adaptation of a euryhaline fish, the sea-bass Dicentrarchus labrax, to various salinities during its ontogeny. The expression and localization of Aquaporin 1a (AQP1a) mRNA and protein were determined in different osmoregulatory tissues. In larvae, the sites of AQP1a expression are variable and they shift according to age, implying functional changes. In juveniles after metamorphosis (D32-D48 post-hatch, 15-25 mm) and in pre-adults, an increase in AQP1a transcript abundance was noted in the digestive tract, and the AQP1a location was observed in the intestine. In juveniles (D87-D100 post-hatch, 38-48 mm), the transcript levels of AQP1a in the digestive tract and in the kidney were higher in sea water (SW) than at lower salinity. These observations, in agreement with existing models, suggest that in SW-acclimated fish, the imbibed water is absorbed via AQP1a through the digestive tract, particularly the intestine and the rectum. In addition, AQP1a may play a role in water reabsorption in the kidney. These mechanisms compensate dehydration in SW, and they contribute to the adaptation of juveniles to salinity changes during sea-lagoon migrations. These results contribute to the interpretation of the adaptation of populations to habitats where salinity varies.
Comparison of amino acid sequences of the AQP1 of silver sea bream (SsAQP1), gilthead sea bream (SaAQP1a), black porgy (AsAQP1), European eel (AaAQP1), human (hsAQP1), and AQP1b of gilthead sea bream (SaAQP1b). Approximate locations of the six transmembrane domains are underlined and two signature NPA motifs are double underlined. Asterisks denote identical amino acid residues among different AQP1 sequences.
Tissue distribution of AQP1a transcript in seawater acclimated silver sea bream. Abbreviations: M, molecular marker (sizes indicated kb); G, gill; H, heart; I, intestine; R, rectum; K, kidney; U, urinary bladder; L, liver; B, brain; and WB, whole blood. A (+) or (−) after an abbreviation indicates reverse transcriptase reactions that were performed with or without MMLV respectively.
Expression of AQP1a transcript in gill (A), intestine (B), and kidney (C) of silver sea bream that were acclimated to freshwater (0 ppt), hypoosmotic (6 ppt), isoosmotic (12 ppt), seawater (33 ppt), hypersaline (50 ppt), and extreme hypersaline (70 ppt) for 1 month. Data is presented as normalized AQP1a transcript abundance (arbitrary units) and values are expressed as mean ± SEM (n = 7). Different letters above a bar denote significant differences among salinity acclimated groups (p < 0.05).
Expression of AQP1a transcript in gill (A), intestine (B), and kidney (C) following an abrupt transfer of silver sea bream. Two transfers were performed, a 33- to 6-ppt abrupt hypoosmotic transfer and a control 33–33 ppt transfer. Data is presented as normalized AQP1a transcript abundance (arbitrary units) and values are expressed as mean ± SEM (n = 7 − 4). There was no significant interaction between salinity and time (p < 0.05).
Expression of AQP1a in gill (A), intestine (B), and kidney (C) of silver sea bream following administration of oil alone (control) or cortisol and then transferred to either 33 or 6 ppt salinity. Data is presented as normalized AQP1a transcript abundance (arbitrary units) and values are expressed as means ± SEM (n = 7). An asterisk above a bar indicates values that were found to be different between oil and cortisol treated groups (p < 0.05).
This study aimed to investigate the effects of chronic salinity acclimation, abrupt salinity transfer, and cortisol administration on aquaporin 1 (AQP1) expression in gill, intestine, and kidney of silver sea bream (Sparus sarba). An AQP1a cDNA was cloned and found to share 83-96% amino acid sequence identity with AQP1 genes from several fish species. Tissue distribution studies of AQP1a mRNA demonstrated that it was expressed in gill, liver, intestine, rectum, kidney, heart, urinary bladder, and whole blood. Semi-quantitative RT-PCR analysis was used to measure AQP1a transcript abundance in sea bream that were acclimated to salinity conditions of 0, 6, 12, 33, 50, and 70 ppt for 1 month. The abundance of gill AQP1a transcript was highest in sea bream acclimated to 0 ppt whereas no differences were found among 0-50 ppt groups. For intestine, the highest AQP1a transcript amounts were found in sea bream acclimated to 12 and 70 ppt whereas the transcript abundance of kidney AQP1a was found to be unchanged amongst the different salinity groups. To investigate the effects of acute salinity alterations on AQP1a expression, sea bream were abruptly transferred from 33 to 6 ppt. For intestine AQP1a levels were altered at different times, post transfer, but remained unchanged in gill and kidney. To study the effects of cortisol on AQP1a expression, sea bream were administered a single dose of cortisol followed by a 3-day acclimation to either 33 or 6 ppt. The findings from this experiment demonstrated that cortisol administration resulted in alterations of AQP1a transcript in gill and intestine but not in kidney.
The cross-correlation cube. The asymptotic cross-correlation function defined by Eq. 3 is graphed as a function of the two power–law indices of the perturbed network S and the perturbing network P. (From West and Grigolini, 2011, with permission.)
(A) Temporal evolution of a single unit and (B) of the global order parameter for the DMM realized on a square lattice with L = 50, g0 = 0.01, and K = 1.70. Notice the different time scales on the two plots. (From Turalska et al., 2011, with permission.)
The phase diagram for the global variable ξeq. The thin solid line and dashed line are theoretical predictions for the fully connected and 2D regular networks, respectively. In both cases L = ∞ and the latter case is the Onsager prediction for a 2D regular lattice. The thick solid line corresponds to the global states observed for a 2D regular lattice with L = 100 and g0 = 0.01. Periodic boundary conditions were applied. (From Turalska et al., 2011, with permission.)
Survival probability function Ψ(τ) for the global order parameter evaluated on a 2D lattice with L = 50, g0 = 0.01 and increasing values of the coupling constant K. The straight line corresponds to a slope of −0.50, namely μ = 1.5 since Ψ(τ)∝1/τμ − 1. (From Turalska et al., 2011, with permission.)
The Army Research Laboratory program on the Network Science of Human Decision Making brings together researchers from a variety of disciplines to work on a complex research problem that defies confinement within any single discipline. Consequently, new and rewarding solutions have been obtained for a problem of importance to society and the Army, that being, the human dimension of complex networks. This program investigates the basic research foundation of a science of networks supporting the linkage between the cognitive and social domains as they relate to human decision making. The research strategy extends recent methods of non-equilibrium statistical physics to non-stationary, renewal stochastic processes characteristic of the interactions among nodes in complex networks. The theoretical analyses of complex networks, although mathematically rigorous, often elude analytic solutions and require simulation and computation to analyze the underlying dynamic process. The information transfer between two complex networks is calculated using the principle of complexity management as well as direct numerical calculation of the decision making model developed within the project.
Visual representation of keywords provided by meeting participants to describe their research area of interest. The weighted list was generated using Word Cloud.
Over the past decade or two biology has become increasingly quantitative and concepts from mathematical and physical sciences have in turn increasingly influenced biology (Knight, 2002; May, 2004; Endy, 2005; Chuang et al., 2010). As a result of these advances in quantitative biology, new biological phenomena at mesoscopic and microscopic scale have been unraveled and techniques to address long-standing fundamental questions have been developed. Yet quantitative biology as a field is reliant on concepts borrowed from engineering and physics and hence is critically dependent on close interaction between theoretical and experimental biologists. The report reviews the highlights of the interactive workshop, and proposes the long-term benefits of such small-scale cross-disciplinary workshops.
Simulation of the Khoury et al. (2011) population dynamic model as implemented by Cresswell and Thompson (2012)2, model parameters were w = 27.000, L = 2000, α = 0.25, σ = 0.75 [as per Henry et al. (2012)], starting hive population was 18,000 (lower bound) or 15,000 (upper bound), pesticide exposure condition assumed the extreme case of 100% foragers exposed. The shaded area corresponds to the exposure period. In blue the projected population growth under a non exposure scenario, in green and red the projected population dynamic under assumptions (a) and (b), respectively, based on Henry et al. (2012) experimental results. Black continuous and dotted line show model projection under assumption (a) and (b) respectively, assuming a normal homing failure of 0.154−1, based on experimental results showing 0.7 and 0.42−1 homing success in the control and treatment respectively (starting population of 18000 individuals).
[Extract] In March 2012 Henry et al. published a paper that explored whether or not the consumption of thiamethoxam via nectar could be a causal factor of Colony Collapse Disorder (CCD) in honeybees. In the first part of their report, Henry et al. (2012) measured the homing success after "ecologically relevant" thiamethoxam exposure and compared it to non-thiamethoxam exposed, control homing rates [see Guez (2013) for a critique of this aspect of their work]. In the second part of their report, they applied their homing study results to the honeybee population dynamic model devised by Khoury et al. (2011), and from that they concluded that dietary thiamethoxam intoxication may potentially contribute to CCD. Khoury et al.'s (2011) model is build upon the hypothesis that colony failure occurs when bee death rate become unsustainable at the colony level, and the salient assumption that mortality within the hive is negligible. Khoury et al.'s (2011) model allows the evolution of the honeybee hive population to be projected over time. Model outputs are dependent on the total hive population (at the start, and then at any given time), the queen's egg laying rate (L), an eclosion rate that is directly dependent upon the hive population and modulated via the parameter w (the larger w the lower the eclosion rate), and the forager mortality rate or forager homing failure (m). In Khoury et al.'s (2011) model therefore, the population growth of the colony is controlled mainly by the parameters L and w [but see Cresswell and Thompson (2012) for a critique of the choice of w], whereas population decline is dependent on m, the forager mortality rate or forager homing failure.
The “Advances in Skeletal Muscle Biology in Health and Disease” conference was held in Gainesville, Florida February 22nd to 24th 2012. The primary purpose of this meeting was to facilitate advances in adult skeletal muscle biology and physiology. There were 210 registered participants from 64 different institutions and 4 pharmaceutical companies, including participants from 10 different countries. Approximately 40% of the attendees and one-third of the symposium speakers were women. The meeting began with a grant writing workshop that included an overview of training grants, R01 grant structure, and grants scoring and interpreting reviewer comments given by Drs. Tom Clanton, Denis Guttridge, Marcas Bamman, and Sue Bodine. This workshop was followed by a poster session that included 65 outstanding posters presented by faculty, postdoctoral fellows, and graduate students. The symposium consisted of 28 total speakers across four main sessions. In this two part synopsis we outline some of the main findings, current paradigms, and future directions presented in each of these sessions. In Part 1, we outline Day 1 of the symposium which consisted of sessions on “Cell signaling mechanisms mediating muscle atrophy and hypertrophy” and “Muscle force, calcium handling, and stress response.” In Part 2, we outline Day 2 of the symposium which consisted of sessions on “Muscle diseases and regeneration” and “Clinical/translational research.”
I have been involved in a publication showing that sublethal exposures to thiamethoxam, a neonicotinoid pesticide, increase the risk of homing failure in foraging honeybees (Henry et al., 2012a). Along with other recent toxicological studies on free-ranging bees (Gill et al., 2012; Schneider et al., 2012; Whitehorn et al., 2012), those results have motivated the European Food Safety Agency (EFSA, 2012) to reconsider the relevance of risk assessment of plant protection products on bees, currently based on the sole lethality criterion. Among the potential indicators of sublethal hazard, homing failure is a suitable candidate (EFSA, 2013) because (i) it integrates multiple physiological and cognitive functions such as orientation, spatial memory, associative learning and muscular flight activity, and (ii) it may be straightforwardly converted into a mortality rate. Homing failure studies are now being replicated for extending the study of behavioral impairments to other substances (Matsumoto, 2013) and even to pathogens (Li et al., 2013). The forthcoming expansion of homing studies in bees underscores the need to set standards for homing failure measurement. In our original contribution (Henry et al., 2012a), we calculated mortality due to homing failure, mhf, as the proportion of nonreturning treated foragers relative to expectations given by the proportion of returning control foragers. This relative control-treatment homing difference returns a mortality probability equivalent to the statistical effect size of the exposure. However, Guez (2013a) criticized our calculation of mhf, arguing that dividing the homing difference by control expectations falsely inflates the mortality estimates. Instead, he claimed mhf should simply read as the absolute control-treatment homing difference. Beside our in-depth reply, alerting on the necessity to properly fix experimental biases (Henry and Decourtye, 2013), Guez (2013b) persisted in his criticism. As a surrogate for our relative homing difference formula [Equation 1 in Guez (2013b)], he recommends either the use of his absolute homing difference formula [Equation 2 in Guez (2013b)] or an alternative formula measuring the proportional increase in post-exposure homing failure [Equation 3 in Guez (2013b)]. I show here that both alternatives are intractable and cannot be properly implemented in a honeybee population dynamics model (Khoury et al., 2011) as in our original study (Henry et al., 2012a). I aim to resolve the disagreement by clarifying several key features of the population models involved. I understand that Guez (2013a,b) implicitly assumes that homing experiments are based on the same temporal scale than the parameters of the honeybee population dynamics model (Khoury et al., 2011). This, however, is incorrect, as shown below. His tentative Equation 2 underestimates mortality, while Equation 3 may return severely overestimated mortalities. I hope this cautionary note will help risk assessors identifying some important pitfalls and challenges in the assessment of post-exposure homing failure in bees.
Copy number analysis of Chromosome 21. (A) Representation of genomic gain (blue) and deletion (red) frequencies on four different tumor types and median average of all tumors together. (B) Deletion frequency for the three major deleted regions (MDR) in breast cancer (BC), melanoma (ML), lung tumors (LT), and Wilms Tumor (WT) on HSA21. (C) Representation of the three MDR based on genomic localization and size with those genes and miRNAs that showed downregulation in the gene expression correlation analysis.
Down syndrome (DS), one of the most common birth defects and the most widespread genetic cause of intellectual disabilities, is caused by extra genetic material on chromosome 21 (HSA21). The increased genomic dosage of trisomy 21 is thought to be responsible for the distinct DS phenotypes, including an increased risk of developing some types of childhood leukemia and germ cell tumors. Patients with DS, however, have a strikingly lower incidence of many other solid tumors. We hypothesized that the third copy of genes located in HSA21 may have an important role on the protective effect that DS patients show against most types of solid tumors. Focusing on Copy Number Variation (CNV) array data, we have generated frequencies of deleted regions in HSA21 in four different tumor types from which DS patients have been reported to be protected. We describe three different regions of deletion pointing to a set of candidate genes that could explain the inverse comorbidity phenomenon between DS and solid tumors. In particular we found RCAN1 gene in Wilms tumors and a miRNA cluster containing miR-99A, miR-125B2 and miR-LET7C in lung, breast, and melanoma tumors as the main candidates for explaining the inverse comorbidity observed between solid tumors and DS.
| Baseline clinical characteristics and demographics.
Effect of renal sympathetic denervation (RDN) on blood pressure (BP) variability and instability: standard deviation of systolic (A) and diastolic (B) BP, maximum systolic (C) and diastolic (D) BP and maximum difference of systolic (E) and diastolic (F) BP observed between two consecutive readings before renal sympathetic denervation and 6 months thereafter.
Effect of renal sympathetic denervation (RDN) on mean levels of blood pressure (BP): office systolic (A) and diastolic (B) BP as well as mean levels of systolic (C) and diastolic (D) blood BP on 24-h ambulatory BP monitoring (ABPM) before renal sympathetic denervation and 6 months thereafter.
Background: In patients with arterial hypertension, increased blood pressure (BP) variability contributes to end organ damage independently from mean levels of arterial BP. Increased BP variability has been linked to alterations in autonomic function including sympathetic overdrive. We hypothesized that catheter-based renal sympathetic denervation (RDN) confers beneficial effects on BP variability. Methods and results: Eleven consecutive patients with therapy-refractory arterial hypertension (age 68.9 ± 7.0 years; baseline systolic BP 189 ± 23 mmHg despite medication with 5.6 ± 2.1 antihypertensive drugs) underwent bilateral RDN. Twenty-four hour ambulatory BP monitoring (ABPM) was performed before RDN and 6 months thereafter. BP variability was primarily assessed by means of standard deviation of 24-h systolic arterial BP (SD(sys)). Secondary measures of BP variability were maximum systolic BP (MAX(sys)) and maximum difference between two consecutive readings of systolic BP (Δmax(sys)) over 24 h. Six months after RDN, SD(sys), MAX(sys), and Δmax(sys) were significantly reduced from 16.9 ± 4.6 to 13.5 ± 2.5 mmHg (p = 0.003), from 190 ± 22 to 172 ± 20 mmHg (p < 0.001), and from 40 ± 15 to 28 ± 7 mmHg (p = 0.006), respectively, without changes in concomitant antihypertensive therapy. Reductions of SD(sys), MAX(sys), and Δmax(sys) were observed in 10/11 (90.9%), 11/11 (100%), and 9/11 (81.8%) patients, respectively. Although we noted a significant reduction of systolic office BP by 30.4 ± 27.7 mmHg (p = 0.007), there was only a trend in reduction of average systolic BP assessed from ABPM (149 ± 19 to 142 ± 18 mmHg; p = 0.086). Conclusion: In patients with therapy-refractory arterial hypertension, RDN leads to significant reductions of BP variability. Effects of RDN on BP variability over 24 h were more pronounced than on average levels of BP.
Ascent profiles of different expeditions and number of AMS cases at different altitudes. Three climbers descended after 4300 m because of AMS. Measurements were made daily up to 5300 m except on Shisha Pangma, where measurements were made up to 5600 m. AMS, Acute Mountain Sickness; AMS*, AMS at this altitude; AMS**, AMS at some other altitude.
Negative correlation between HR and positive correlation between lnHF, lnLF, and RMSSD at 2400 m altitude and the lowest altitude at which a climber got AMS (HR r = −0.743, p < 0.01; lnHF r = 0.558, p < 0.05, lnLF r = 0.302, p > 0.05, RMSSD r = 0.495, p < 0.05). no-AMS subjects datapoints are added at 5600 m level.
| Resting heart rate (HR) and HRV parameters at 2400 m among the climbers who subsequently developed AMS at two different altitude ranges, and those who had no subsequent AMS.
Scattergram showing the distribution of values of RMSSD and Ex-SpO2 measured in 24 study subjects at 2400 m in AMS and no-AMS. The cut-off lines RMSSD2min ≤ 30 ms and Ex-SpO2 ≤ 91% are for 92% sensitivity for AMS.
Objective: If the body fails to acclimatize at high altitude, acute mountain sickness (AMS) may result. For the early detection of AMS, changes in cardiac autonomic function measured by heart rate variability (HRV) may be more sensitive than clinical symptoms alone. The purpose of this study was to ascertain if the changes in HRV during ascent are related to AMS. Methods: We followed Lake Louise Score (LLS), arterial oxygen saturation at rest (R-SpO(2)) and exercise (Ex-SpO(2)) and HRV parameters daily in 36 different healthy climbers ascending from 2400 m to 6300 m altitudes during five different expeditions. Results: After an ascent to 2400 m, root mean square successive differences, high-frequency power (HF(2 min)) of HRV were 17-51% and Ex-SpO(2) was 3% lower in those climbers who suffered from AMS at 3000 to 4300 m than in those only developing AMS later (≥5000 m) or not at all (all p < 0.01). At the altitude of 2400 m RMSSD(2 min) ≤ 30 ms and Ex-SpO(2) ≤ 91% both had 92% sensitivity for AMS if ascent continued without extra acclimatization days. Conclusions: Changes in supine HRV parameters at 2400 m were related to AMS at 3000-4300 m Thus, analyses of HRV could offer potential markers for identifying the climbers at risk for AMS.
Mutations of the GJB2 gene encoding the connexin 26 (Cx26) gap junction protein, which is widely expressed in the inner ear, are the primary cause of hereditary non-syndromic hearing loss in several populations. The deafness-associated single amino acid substitution of methionine 34 (M34) in the first transmembrane helix (TM1) with a threonine (T) ensues in the production of mutant Cx26M34T channels that are correctly synthesized and assembled in the plasma membrane. However, mutant channels overexpressed in HeLa cells retain only 11% of the wild type unitary conductance. Here we extend and rationalize those findings by comparing wild type Cx26 (Cx26WT) and Cx26M34T mutant channels in silico, using molecular dynamics simulations. Our results indicate that the quaternary structure of the Cx26M34T hemichannel is altered at the level of the pore funnel due to the disruption of the hydrophobic interaction between M34 and tryptophan 3 (W3) in the N-terminal helix (NTH). Our simulations also show that external force stimuli applied to the NTHs can detach them from the inner wall of the pore more readily in the mutant than in the wild type hemichannel. These structural alterations significantly increase the free energy barrier encountered by permeating ions, correspondingly decreasing the unitary conductance of the Cx26M34T hemichannel. Our results accord with the proposal that the mutant resides most of the time in a low conductance state. However, the small displacement of the NTHs in our Cx26M34T hemichannel model is not compatible with the formation of a pore plug as in the related Cx26M34A mutant.
Biophysical properties suggest transport of H+ into endosomes by CLC-5. (A) A family of CLC-5 currents recorded from a transfected HEK293 cell by the whole-cell patch-clamp technique. The dotted line represents the zero current baseline. Pulses were applied from −100 to +100 mV as illustrated by the voltage protocol. Only outward currents (upward currents from the baseline), corresponding to H+ efflux and Cl− influx, are observed with CLC-5 expressed at the cell surface, indicated by the arrows to the right of the currents. Ion flux in the opposite direction would be recorded as inward currents (downward deflection from baseline) and these were not observed under all [Cl−] and pH conditions tested (Smith and Lippiat, 2010a). The transient inward currents upon repolarization are gating currents and do not involve ion transport across the membrane (Smith and Lippiat, 2010b). (B) If this property persists in endosomal membranes then a Cl− gradient, which may occur immediately upon endocytic vesicle formation, may drive the transport of H+ into endosomes by CLC-5. It is assumed that the arrangement of transmembrane and intracellular domains of CLC-5 are consistent between plasma and endosomal membranes, as indicated by the representation of the crystal structure of a homologous eukaryotic CLC transporter (Feng et al., 2010). Ion transport through one subunit of the dimer is indicated by the arrows and the proteins and could provides the H+-ATPase-independent acidification component identified by (Smith and Lippiat, 2010a). Other transporters and membrane proteins that colocalize with CLC-5 are omitted from this cartoon.
CLC-5 plays a critical role in the process of endocytosis in the proximal tubule of the kidney and mutations that alter protein function are the cause of Dent's I disease. In this X-linked disorder impaired reabsorption results in the wasting of calcium and low molecular weight protein to the urine, kidney stones, and progressive renal failure. Several different ion-transporting and protein clustering roles have been proposed as the physiological function of CLC-5 in endosomal membranes. At the time of its discovery, nearly 20 years ago, it was understandably assumed to be a chloride channel similar to known members of the CLC family, such as CLC-1, suggesting that chloride transport by CLC-5 was critical for endosomal function. Since then CLC-5 was found instead to be a 2Cl(-)/H(+) exchange transporter with voltage-dependent activity. Recent studies have determined that it is this coupled exchange of protons for chloride, and not just chloride transport, which is critical for endosomal and kidney function. This review discusses the recent ideas that describe how CLC-5 might function in endosomal membranes, the aspects that we still do not understand, and where controversies remain.
Himantura signifer is a freshwater stingray which inhabits rivers in Southeast Asia. It can survive in brackish water but not seawater. In brackish water, it becomes partially ureosmotic, but how it maintains its plasma hypoionic to the external medium is enigmatic because of the lack of a rectal gland. Here, we report for the first time the expression of Na(+):K(+):2Cl(-) cotransporter 1 (nkcc1) in the gills of freshwaterH. signifer, and its moderate up-regulation (~2-fold) in response to brackish water (salinity 20) acclimation. The absence of the Ste20-related proline-alanine-rich kinase and oxidation stress response kinase 1 interaction site from the N-terminus of H. signifer Nkcc1 suggested that it might not be effectively activated by stress kinases in response to salinity changes as in more euryhaline teleosts. The increased activity of Nkcc1 during salt excretion in brackish water would lead to an influx of Na(+) into ionocytes, and the maintenance of intracellular Na(+) homeostasis would need the cooperation of Na(+)/K(+)-ATPase (Nka). We demonstrated for the first time the expression of nkaα1, nkaα2 and nkaα3 in the gills of H. signifer, and the up-regulation of the mRNA expression of nkaα3 and the overall protein abundance of Nkaα in response to acclimation to brackish water. Immunofluorescence microscopy revealed the presence of a sub-type of ionocyte, co-expressing Nkcc1 and Nkaα, near the base of the secondary lamellae in the gills of H. signifer acclimated to brackish water, but this type of ionocyte was absent from the gills of fish kept in fresh water. Hence, there could be a change in the function of the gills of H. signifer from salt absorption to salt excretion during brackish water acclimation in the absence of a functioning rectal gland.
Synoptic of the workflow of the proteomic approaches so far utilized to obtain new information on the actions that T2 and T3 exert in vivo in key metabolically active tissues central in the control of energy balance. The described studies were performed as integrated approaches including 2D-E, mass spectrometry, and bioinformatic tools (Silvestri et al., 2006, 2007, 2010; Moreno et al., 2011). Abbreviations: N, standard diet fed control rats; HFD, high fat diet fed rats; HFD+T2, high fat diet fed rats treated with T2; Eu, euthyroid rats; Hypo, hypothyroid rats; Hypo+T3, hyperthyroid rats.
In vertebrates and, specifically, in mammals, energy homeostasis is achieved by the integration of metabolic and neuroendocrine signals linked to one another in an intricate network hierarchically responding to the tight modulating action of hormones among which thyroid hormones (THs) play a central role. At the cellular level, 3,5,3'-triiodo-L-thyronine (T3) acts mainly by binding to specific nuclear receptors (TRs) but actually it is becoming more and more evident that some T3- actions are independent of TRs and that other iodothyronines, such as 3,5-diiodo-L-thyronine (T2), affect energy metabolism and adiposity. In the postgenomic era, clinical and basic biological researches are increasingly benefiting from the recently developed new omics approaches including, among the others, proteomics. Considering the recognized value of proteins as excellent targets in physiology, the functional and simultaneous analysis of the expression level and the cellular localization of multiple proteins can actually be considered fundamental in the understanding of complex mechanisms such as those involved in thyroid control of metabolism. Here, we will discuss new leads (i.e., target proteins and metabolic pathways) emerging in applying proteomics to the actions of T3 and T2 in conditions of altered energy metabolism in animal tissues having a central role in the control of energy balance.
While the morphology and function of cells of the exocrine and endocrine pancreas have been studied over several centuries, one important cell type in the gland, the pancreatic stellate cell (PSC), had remained undiscovered until as recently as 20 years ago. Even after its first description in 1982, it was to be another 16 years before its biology could begin to be studied, because it was only in 1998 that methods were developed to isolate and culture PSCs from rodent and human pancreas. PSCs are now known to play a critical role in pancreatic fibrosis, a consistent histological feature of two major diseases of the pancreas-chronic pancreatitis and pancreatic cancer. In health, PSCs maintain normal tissue architecture via regulation of the synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies have also implied other functions for PSCs as progenitor cells, immune cells or intermediaries in exocrine pancreatic secretion in humans. During pancreatic injury, PSCs transform from their quiescent phase into an activated, myofibroblast-like phenotype that secretes excessive amounts of ECM proteins leading to the fibrosis of chronic pancreatitis and pancreatic cancer. An ever increasing number of factors that stimulate and/or inhibit PSC activation via paracrine and autocrine pathways are being identified and characterized. It is also now established that PSCs interact closely with pancreatic cancer cells to facilitate cancer progression. Based on these findings, several therapeutic strategies have been examined in experimental models of chronic pancreatitis as well as pancreatic cancer, in a bid to inhibit/retard PSC activation and thereby alleviate chronic pancreatitis or reduce tumor growth in pancreatic cancer. The challenge that remains is to translate these pre-clinical developments into clinically applicable treatments for patients with chronic pancreatitis and pancreatic cancer.
Assessment of tooth morphology is an important part of the diagnosis and management of hypodontia patients. Several techniques have been used to analyze tooth form in hypodontia patients and these have shown smaller tooth dimensions and anomalous tooth shapes in patients with hypodontia when compared with controls. However, previous studies have mainly used 2D images and provided limited information. In the present study, 3D surface-imaging and statistical shape analysis were used to evaluate tooth form differences between hypodontia and control patients. Eighteen anatomical landmarks were recorded on the clinical crown of the lower left first permanent molar of 3D scanned study models of hypodontia and control subjects. The study sample group comprised of 120 hypodontia patients (40 mild, 40 moderate, and 40 severe hypodontia patients) and 40 age- and sex-matched controls. Procrustes coordinates were utilized to scale and superimpose the landmark coordinate data and then were subjected to principal component analysis (PCA). Subsequently, differences in shape as well as size were tested statistically using allometric analysis and MANOVA. Significant interaction was found between the two factor variables "group" and "sex" (p < 0.002). Overall expected accuracies were 66 and 56% for females and males, respectively, in the cross-validated discriminant-analysis using the first 20 PCs. Hypodontia groups showed significant shape differences compared with the control subjects (p < 0.0001). Significant differences in tooth crown shape were also found between sexes (p < 0.0001) within groups. Furthermore, the degree of variation in tooth form was proportional to the degree of the severity of the hypodontia. Thus, quantitative measurement of tooth shape in hypodontia patients may enhance the multidisciplinary management of those patients.
Connexin 43 (Cx43), the principal gap junction protein in vascular smooth muscle cells (VSMCs), regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC) and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY), or the Cx inducer diallyl disulfide (DADS) significantly reduced proliferation after 72 h compared with vehicle controls. Bromodeoxyuridine uptake revealed reduction (p < 0.05) in DNA synthesis after 6 h and flow cytometry showed reduced (40%) S-phase cell numbers after 16 h in DADS-treated cells compared with vehicle controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at MAPK-sensitive Serine (Ser)255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared with controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays important roles in the regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSM.
Cell-to-cell interactions among osteoclasts, osteoblasts, and osteocytes in close proximity maintain bone homeostasis. (A) An osteoclast (Oc), a team of osteoblasts (Ob) and bone-embedded osteocytes (Ot) are shown in a rat bone section, in which osteoclasts were stained for the osteoclast-specific enzyme tartrate resistant acid phosphatase (TRAPase) (red) and counterstained using Toluidine blue. Scale bar indicates 50 μm. Picture contributed by Keith W. Condon (Indiana University School of Medicine). (B) Transmission electron microscope image of an osteocyte embedded in the bone matrix. Image was obtained at the Electron Microscopy Center of the Department of Anatomy and Cell Biology (Indiana University School of Medicine). Scale bar indicates 5 μm (C) Scanning electron microscope image of an acid-etched bone showing two osteocytes highly communicated through their cytoplasmic projections (Bellido et al., 2014). Scale bar indicates 1 μm. (D) Cx43 immunostaining of a section of cancellous bone showing osteoblasts (Ob) on the bone surface and osteocytes (Ot) embedded in the bone matrix stained for Cx43 (brown) and counterstained with Methyl green (Plotkin et al., 2008). Scale bar indicates 50 μm.
Schematic representation of the proposed intracellular signaling pathways regulated by Cx43 hemichannels in bone cells. (A) Bisphosphonates bind to phosphatases present in the cell membrane. This induces Cx43 hemichannel opening, followed by activation of the kinases Src and MEK, and ERKs. ERKs activated downstream of Cx43 hemichannel opening are retained in the cytoplasm by a complex formed by βarrestin and clathrins. This leads to the phosphorylation of the cytoplasmic targets p90RSK, BAD and C/EBPβ, which results in osteoblast and osteocyte survival. (B) Mechanical stimulation induces α5β1 integrin engagement and the association of the integrins with Cx43, by a mechanism that requires the protein 14-3-3θ. This results in hemichannel opening and the release of PGE2. PGE2, in turn, activates EP2/4 prostaglandin receptor by an autocrine/paracrine mechanism, leading to activation of the cAMP/PI3K signaling pathways, accumulation of βcatenin with the consequent activation of Wnt signaling, and inhibition of osteocyte apoptosis. (C) Parathyroid hormone (PTH), through binding to the PTH receptor, induces activation of the cAMP/PKA signaling pathway. Cx43, by sequestering β-arrestin away from the PTH receptor, facilitate cAMP/PKA-mediated downstream signaling and osteoblast survival.
Cell function and survival are controlled by intracellular signals, and modulated by surrounding cells and the extracellular environment. Connexin channels participate in these processes by mediating cell-to-cell communication. In bone cells, gap junction channels were detected in the early 1970s, and are present among bone resorbing osteoclasts, bone forming osteoblasts, and osteocytes - mature osteoblasts embedded in the mineralized matrix. These channels are composed mainly by Cx43, although the expression of other connexins (45, 46, and 37) has also been reported. It is now believed that undocked Cx43 hemichannels (connexons) formed in unopposed cell membranes facing the extracellular environment participate in the interaction of bone cells with the extracellular environment, and in their communication with neighboring cells. Thus, we and others demonstrated the presence of active hemichannels in osteoblastic and osteocytic cells. These hemichannels open in response to pharmacological and mechanical stimulation. In particular, preservation of the viability of osteoblasts and osteocytes by the anti-osteoporotic drugs bisphosphonates depends on Cx43 expression in vitro and in vivo, and is mediated by undocked hemichannels. Cx43 hemichannels are also required for the release of prostaglandins and ATP by osteocytes, and for cell survival induced by mechanical stimulation in vitro. Moreover, they are required for the anti-apoptotic effect of parathyroid hormone in osteoblastic cells. This review summarizes the current knowledge on the presence and function of undocked connexons, and the role of hemichannel regulation for the maintenance of bone cell viability and, potentially, bone health.
The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca(2+) into cells and to the Ca(2+)-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of (45)Ca(2+) uptake and [(14)C]-methyl aminoisobutyric acid ([(14)C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca(2+) channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of (45)Ca(2+) entry and [(14)C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca(2+) channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I.
Background: MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. miRNAs are taken in by intracellular exosomes, secreted into circulation, and taken up by other cells, where they regulate cellular functions. We hypothesized that muscle-enriched miRNAs existing in circulation mediate beneficial metabolic responses induced by exercise. To test this hypothesis, we measured changes in muscle-enriched circulating miRNAs (c-miRNAs) in response to acute and chronic aerobic exercise. Methods: Eleven healthy young men (age, 21.5 ± 4.5 y; height, 168.6 ± 5.3 cm; and body weight, 62.5 ± 9.0 kg) performed a single bout of steady-state cycling exercise at 70% VO2max for 60 min (acute exercise) and cycling training 3 days per week for 4 weeks (chronic exercise). Blood samples were collected from the antecubital vein before and after acute and chronic exercise. RNA was extracted from serum, and the levels of muscle-enriched miRNAs (miR-1, miR-133a, miR-133b, miR-206, miR-208b, miR-486, and miR-499) were measured. Results: All of these miRNAs, except for miR-486, were found at too low copy numbers at baseline to be compared. miR-486 was significantly decreased by both acute (P = 0.013) and chronic exercise (P = 0.014). In addition, the change ratio of miR-486 due to acute exercise showed a significant negative correlation with VO2max for each subject (R = 0.58, P = 0.038). Conclusion: The reduction in circulating miR-486 may be associated with metabolic changes during exercise and adaptation induced by training.
Background: The angiotensin converting enzyme 2 (ACE2) G8790A gene polymorphism has been associated with the susceptibility to essential hypertension (EH), but the results are disputable. Objective and methods: To investigate the relationship between the ACE2 G8790A gene polymorphism and EH, eight separate studies with 5260 subjects were meta-analyzed. The pooled odds ratio (OR) and its corresponding 95% confidence interval (CI) were calculated by a random effect model. Results: In the ACE2 G8790A gene polymorphism and EH meta-analysis in a Chinese population, no significant association was found between the ACE2 G8790A gene polymorphism and EH (OR: 1.03, 95% CI: 0.87-1.21, P = 0.76). In the stratified analysis by gender, no significant risk was found among males (OR: 1.06, 95% CI: 0.82-1.36, P = 0.66) or females (OR: 0.98, 95% CI: 0.77-1.24, P = 0.85). Under a dominant model of inheritance in the female subgroup, the pooled OR for the GG/GA + AA value was 1.01 (95% CI: 0.82-1.25, P = 0.92). Under a recessive model of inheritance in the female subgroup, the pooled OR for the AA/AG + GG value was 0.93 (95% CI: 0.50-1.73, P = 0.83). Conclusion: The current meta-analysis suggested that the ACE2 G8790A gene polymorphism might not be related to the increased EH risk in the Chinese population.
| List of studies which have directly manipulated Hsp70 expression to investigate the functions of Hsp70 in regulating skeletal muscle plasticity. 
The stress-inducible 70-kDa heat shock protein (HSP70) is a highly conserved protein with diverse intracellular and extracellular functions. In skeletal muscle, HSP70 is rapidly induced in response to both non-damaging and damaging stress stimuli including exercise and acute muscle injuries. This upregulation of HSP70 contributes to the maintenance of muscle fiber integrity and facilitates muscle regeneration and recovery. Conversely, HSP70 expression is decreased during muscle inactivity and aging, and evidence supports the loss of HSP70 as a key mechanism which may drive muscle atrophy, contractile dysfunction and reduced regenerative capacity associated with these conditions. To date, the therapeutic benefit of HSP70 upregulation in skeletal muscle has been established in rodent models of muscle injury, muscle atrophy, modified muscle use, aging, and muscular dystrophy, which highlights HSP70 as a key therapeutic target for the treatment of various conditions which negatively affect skeletal muscle mass and function. This article will review these important findings and provide perspective on the unanswered questions related to HSP70 and skeletal muscle plasticity which require further investigation.
α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components.
| ABCA1/ABCB1 Transport Systems in the NVU: Proposed mechanism via which ABCA1/ApoE-LRP-1/ABCB1 systems in the NVU cooperate in order to efficiently mediate Aβ clearance from brain parenchyma, and microvessels into blood circulation. ABCA1 in brain
ABCA1/ABCB1 Transport Systems in the NVU: Proposed mechanism via which ABCA1/ApoE—LRP-1/ABCB1 systems in the NVU cooperate in order to efficiently mediate Aβ clearance from brain parenchyma, and microvessels into blood circulation. ABCA1 in brain endothelial cells, pericytes, astrocytes, and microglial cells enhances ApoE lipidation, and with the collaboration of ABCB1 facilitates Aβ trafficking into the perivascular space, where it can be subsequently eliminated into blood circulation via LRP-1/ABCB1 in brain endothelial cells.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects elderly persons, evolving with age to reach severe cognitive impairment. Amyloid deposits and neurofibrillary tangles constitute the main pathological hallmarks of AD. Amyloid deposits are initiated by the excessive production and accumulation of beta-amyloid (Aβ) peptides in the brain. The dysfunction of the Neurovascular Unit (NVU) has been proposed to be causative in AD development, due to an impaired clearance of Aβ from the brain. Cells forming the NVU express several Adenosine Triphosphate ATP-Binding Cassette (ABC) transporters, among which ABCB1 and ABCA1 play an important role in Aβ processing. The drug transporter ABCB1 directly transports Aβ from the brain into the blood circulation, whereas the cholesterol transporter ABCA1 neutralizes Aβ aggregation capacity in an Apolipoprotein E (ApoE)-dependent manner, facilitating Aβ subsequent elimination from the brain. In the present minireview, we will summarize the contribution of ABCB1, and ABCA1 at the NVU in Aβ clearance. Moreover, we will outline and discuss the possible collaboration of ABCB1, and ABCA1 at the NVU in mediating an efficient clearance of Aβ from the brain.
Abdominal Aortic Aneurysms (AAAs) are frequently characterized by the presence of an Intra-Luminal Thrombus (ILT) known to influence their evolution biochemically and biomechanically. The ILT progression mechanism is still unclear and little is known regarding the impact of the chemical species transported by blood flow on this mechanism. Chemical agonists and antagonists of platelets activation, aggregation, and adhesion and the proteins involved in the coagulation cascade (CC) may play an important role in ILT development. Starting from this assumption, the evolution of chemical species involved in the CC, their relation to coherent vortical structures (VSs) and their possible effect on ILT evolution have been studied. To this end a fluid-chemical model that simulates the CC through a series of convection-diffusion-reaction (CDR) equations has been developed. The model involves plasma-phase and surface-bound enzymes and zymogens, and includes both plasma-phase and membrane-phase reactions. Blood is modeled as a non-Newtonian incompressible fluid. VSs convect thrombin in the domain and lead to the high concentration observed in the distal portion of the AAA. This finding is in line with the clinical observations showing that the thickest ILT is usually seen in the distal AAA region. The proposed model, due to its ability to couple the fluid and chemical domains, provides an integrated mechanochemical picture that potentially could help unveil mechanisms of ILT formation and development.
| CB modeling of energy metabolism. Constraints and condition-specific parameters are most commonly represented as bounds on the flux through reactions in the network; optimizing for specific cellular objectives identifies network states that satisfy
CB modeling of energy metabolism. Constraints and condition-specific parameters are most commonly represented as bounds on the flux through reactions in the network; optimizing for specific cellular objectives identifies network states that satisfy constraints and simulate in vivo or in vitro metabolism. Computational tools can be used to analyze models and investigate metabolic capabilities. The center panel highlights important components of cellular energy metabolism.
Schematic of the major metabolic processes housed in the mitochondria, and detail of the TCA cycle and the GABA shunt (GABA shunt related reactions in purple). AcCoA, acetyl coenzyme A; ADP, adenosine diphosphate; aKG, α-ketoglutarate; ATP, adenosine triphosphate; Cit, citrate; Fum, fumarate; GABA, γ-aminobutyric acid; Glu, glutamate; iCit, isocitrate; Mal, malate; NADH, nicotamide adenine dinucleotide; OA, oxaloacetate; Pyr, pyruvate; Succ, succinate; SuccCoA, succinyl-coenzyme A; Sucsal, succinate semialdehyde.
Future areas of focus for CB modeling of mitochondrial metabolism. Depicted here are several potential research areas that will be explored in future modeling endeavors, including the effects of (1) metabolite exchange between mitochondria and other compartments; (2) dynamic mitochondrial populations, in terms of size and age; and (3) free radical induced mutations in mitochondrial DNA.
Dysfunction in energy metabolism-including in pathways localized to the mitochondria-has been implicated in the pathogenesis of a wide array of disorders, ranging from cancer to neurodegenerative diseases to type II diabetes. The inherent complexities of energy and mitochondrial metabolism present a significant obstacle in the effort to understand the role that these molecular processes play in the development of disease. To help unravel these complexities, systems biology methods have been applied to develop an array of computational metabolic models, ranging from mitochondria-specific processes to genome-scale cellular networks. These constraint-based (CB) models can efficiently simulate aspects of normal and aberrant metabolism in various genetic and environmental conditions. Development of these models leverages-and also provides a powerful means to integrate and interpret-information from a wide range of sources including genomics, proteomics, metabolomics, and enzyme kinetics. Here, we review a variety of mechanistic modeling studies that explore metabolic functions, deficiency disorders, and aberrant biochemical pathways in mitochondria and related regions in the cell.
| Comparison of monitoring devices for atrial fibrillation. 
Atrial fibrillation (AF) is the most common arrhythmia prompting clinical presentation, is associated with significant morbidity and mortality. The incidence and prevalence of this arrhythmia is expected to grow significantly in the coming decades. Of the available pharmacologic and non-pharmacologic treatment options, the fastest growing and most intensely studied is catheter-based ablation therapy for AF. Given the varying success rates for AF ablation, the increasingly complex factors that need to be taken into account when deciding to proceed with ablation, as well as varying definitions of procedural success, accurate detection of arrhythmia recurrence and its burden is of significance. Detecting and monitoring AF recurrence following catheter ablation is therefore an important consideration. Multiple studies have demonstrated the close relationship between the intensity of rhythm monitoring with wearable ambulatory cardiac monitors, or implantable cardiac rhythm monitors and the detection of arrhythmia recurrence. Other studies have employed algorithms dependent on intensive monitoring and arrhythmia detection in the decision tree on whether to proceed with repeat ablation or medical therapy. In this review, we discuss these considerations, types of monitoring devices, and implications for monitoring AF recurrence following catheter ablation.
Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca2+-sensitivity (pCa50) and length-dependent changes in kinetic parameters of cross-bridge (XB) relaxation (krel), and recruitment (kdf) due to rapid stretch, as well as the rate of force redevelopment (ktr) in response to a large slack-restretch maneuver in skinned ventricular multicellular preparations isolated from the hearts of wild-type (WT) and cMyBP-C knockout (KO) mice, at SL’s 1.9µm or 2.1µm. Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82±0.02 in WT vs. 5.87±0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL (5.71±0.02 in WT vs. 5.80±0.01 in KO; p<0.05). The ktr, measured at half-maximal Ca2+-activation, was significantly accelerated at short SL in WT preparations (8.74±0.56s-1at 1.9µm vs. 5.71±0.40s-1at 2.1µm, p<0.05). Furthermore, krel and kdf were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, ktr was not altered by changes in SL in KO preparations (8.03±0.54s-1at 1.9µm vs. 8.90±0.37s-1at 2.1µm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C is an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.
Abnormal ventricular wall motion is a strong clinical predictor of sudden, arrhythmic, cardiac death. Dispersion in repolarization is a prerequisite for the initiation of re-entrant arrhythmia. We hypothesize that regionally decreased wall motion is associated with heterogeneity of repolarization. We measured local activation times, activation-recovery intervals (ARIs, surrogate for action potential duration), and repolarization times using a multielectrode grid at nine segments on the left ventricular epicardium in 23 patients undergoing coronary artery surgery. Regional wall motion was simultaneously assessed using intraoperative transesophageal echocardiography. Three groups were discriminated: (1) Patients with normal wall motion (n = 11), (2) Patients with one or more hypokinetic segments (n = 6), (3) Patients with one or more akinetic or dyskinetic segments (n = 6). The average ARI was similar in all groups (251 ± 3.7 ms, ±SEM). Dispersion of ARIs between the nine segments was significantly increased in the hypokinetic (84 ± 7.4 ms, p < 0.005) and akinetic/dyskinetic group (94 ± 3.5 ms, p < 0.0005) compared with the normal group (49 ± 5.1 ms), independent from the presence of myocardial infarction. Repolarization heterogeneity occurred primarily in the normally contracting regions of the hearts with abnormal wall motion. An almost maximal increased dispersion of repolarization was observed when there was only a single hypokinetic segment. We conclude that inhomogeneous wall motion abnormality of even moderate severity is associated with increased repolarization inhomogeneity, independent from the presence of infarction.
Frontal bone development in Gli3Xt-J/Xt-J mice. Alcian blue and Alizarin red S stained heads of Wt (A,C,F,K,P) and Gli3Xt-J/Xt-J
(B,D,G–J,L–O,Q–S) embryos and a schematic of Gli3Xt-J/Xt-J head indicating heterotopic ossification (red), heterotopic cartilage formation (blue), and measurements of the head (green arrows) (E). At E18.5 Gli3Xt-J/Xt-J frontal bones are abnormally shaped (black arrow in H) and in the interfrontal suture there are heterotopic bones [(G,H); double-lined arrow] that have fused in some samples causing suture synostosis. In samples with less heterotopic bones the suture is wider compared to Wt [(I); arrowhead]. At E17.5 Gli3Xt-J/Xt-J frontal bones have similar features as at E18.5. At E16.5 Gli3Xt-J/Xt-J frontal bones are hypoplastic, but ectopic ossification is already evident. Ectopic cartilage is seen in the interfrontal suture of Gli3Xt-J/Xt-J mice [(I,J,L,N,O,Q,R,U); green arrow]. Toluidine blue stained frontal sections through the posterior interfrontal suture of Wt (T) and Gli3Xt-J/Xt-J
(U) heads at E16.5. Morphology of the Gli3Xt-J/Xt-J brain is abnormal (U). Dash line in T and U indicate the midline of the head. Alkaline phosphatase stained E13.5 heads of Wt (V,X) and Gli3Xt-J/Xt-J
(W,Y,Z) embryos, where heterotopic osteoblast differentiation is detected in Gli3Xt-J/Xt-J mice [(W,Y,Z); red arrow]. f, Frontal bone; i, interparietal bone; is, interfrontal suture; ls, lambdoid suture; p, parietal bone; ss, sagittal suture. Scale bars: 2 mm, except 300 μm in (T) and 500 μm in (X,Z).
Osteoblast differentiation and brain morphology of Gli3Xt-J/Xt-J mice at E16.5. Frontal sections of Wt (A,C) and Gli3Xt-J/Xt-J heads (B,D). (A–D) The osteoblastic markers Runx2 and Bsp are expressed in the frontal bones and ectopically in the interfrontal suture (arrows) of Gli3Xt-J/Xt-J mice. Dash line in (E,F) represent the plane of section in (A–D) respectively. Brains of Wt (E,G) and Gli3Xt-J/Xt-J
(F,H) mice. The Gli3Xt-J/Xt-J brain has hypoplastic dorsal telencephalon and diencephalon resides more anteriorly. The cerebellum extends more ventrally. cb, Cerebellum; dc, diencephalon, f, frontal; is, interfrontal suture; e, eye; mb, midbrain; ob, olfactory bulb; tc, telencephalon. Scale bars are 1 mm in (A), 2 mm in (E).
Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder with polydactyly and syndactyly of the limbs and a broad spectrum of craniofacial abnormalities. Craniosynostosis of the metopic suture (interfrontal suture in mice) is an important but rare feature associated with GCPS. GCPS is caused by mutations in the transcription factor GLI3, which regulates Hedgehog signaling. The Gli3 loss-of-function (Gli3(Xt-J/Xt-J)) mouse largely phenocopies the human syndrome with the mice exhibiting polydactyly and several craniofacial abnormalities. Here we show that Gli3(Xt-J/Xt-J) mice exhibit ectopic ossification in the interfrontal suture and in the most severe cases the suture fuses already prior to birth. We show that abnormalities in frontal bones occur early in calvarial development, before the establishment of the interfrontal suture. It provides a model for the metopic suture pathology that can occur in GCPS.
| Summary of RyR2 mutations associated with atrial and/or sinoatrial node dysfunction.
(A) Atrial monophasic action potential recording of arrhythmic events provoked by programmed electrical stimulation involving extrasystolic S2 stimulation (long vertical bars beneath trace) following series of pacing S1 stimuli (short vertical bars beneath trace) from isolated perfused homozygotic RyR2-P2328S heart. (B) Ca2+ transients from regularly stimulated fluo-3-loaded RyR2-P2328S atrial myocytes under confocal microscopy, both following introduction of isoproterenol [From Figures 5D, 6F of Zhang et al. (2011)].
Ryanodine receptor type 2 (RyR2) mutations are implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT) thought to result from altered myocyte Ca(2+) homeostasis reflecting inappropriate "leakiness" of RyR2-Ca(2+) release channels arising from increases in their basal activity, alterations in their phosphorylation, or defective interactions with other molecules or ions. The latter include calstabin, calsequestrin-2, Mg(2+), and extraluminal or intraluminal Ca(2+). Recent clinical studies additionally associate RyR2 abnormalities with atrial arrhythmias including atrial tachycardia (AT), fibrillation (AF), and standstill, and sinus node dysfunction (SND). Some RyR2 mutations associated with CPVT in mouse models also show such arrhythmias that similarly correlate with altered Ca(2+) homeostasis. Some examples show evidence for increased Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2. A homozygotic RyR2-P2328S variant demonstrates potential arrhythmic substrate resulting from reduced conduction velocity (CV) in addition to delayed afterdepolarizations (DADs) and ectopic action potential (AP) firing. Finally, one model with an increased RyR2 activity in the sino-atrial node (SAN) shows decreased automaticity in the presence of Ca(2+)-dependent decreases in I Ca, L and diastolic sarcoplasmic reticular (SR) Ca(2+) depletion.
B-mode ultrasound image from a live-stranded common dolphin (Delphinus delphis). Reverberation artifact of normal lung. Some of the liver is seen on the left side of the images. The gas within the periphery of the lung causes near perfect reflection of the sound beam. The result is a repeated, equally spaced hyperechoic line (bright) being displayed on the image (arrows).
B-mode ultrasound image of a live-stranded common dolphin (Delphinus delphis) kidney. Multiple hyperechoic (bright) foci are observed at the top of the image (black arrow). Ring-down artifact is seen (white arrows) as repeating hyperechoic (bright) lines that do not taper or diminish toward the bottom of the image, confirming that gas is present.
A CT image of the head at the level of the eyes from a bycatch gray seal (Halichoerus grypus). The image was acquired using a bone reconstruction algorithm and displayed on a wide window that results in only gas being displayed as dark gray or black. Normal gas accumulations in the nasopharynx (*) and oral cavity (white circle) are observed. Abnormal gas accumulations within tissues and vasculature, some demonstrated by black arrows, are also present.
Magnetic resonance imaging images from a California sea lion (Zalophus californianus) to demonstrate the appearance of gas and the associated susceptibility artifact. Both images are at the same level on the same patient. T2W is a regular MRI sequence and T2*W is sensitive to susceptibility artifact. The air within the bulla (*) on the T2W image can be seen and the inner ear structures (arrow) are also present. The same image is shown after T2*W acquisition and the amount of gas within and overall size of the bulla looks larger and the “blooming” effect of the susceptibility artifact due to an air-tissue interface results in loss of conspicuity of the inner ear structures. If abnormal gas were present in the inner ear, it would not be identifiable from this image.
Recent dogma suggested that marine mammals are not at risk of decompression sickness due to a number of evolutionary adaptations. Several proposed adaptations exist. Lung compression and alveolar collapse that terminate gas-exchange before a depth is reached where supersaturation is significant and bradycardia with peripheral vasoconstriction affecting the distribution, and dynamics of blood and tissue nitrogen levels. Published accounts of gas and fat emboli and dysbaric osteonecrosis in marine mammals and theoretical modeling have challenged this view-point, suggesting that decompression-like symptoms may occur under certain circumstances, contrary to common belief. Diagnostic imaging modalities are invaluable tools for the non-invasive examination of animals for evidence of gas and have been used to demonstrate the presence of incidental decompression-related renal gas accumulations in some stranded cetaceans. Diagnostic imaging has also contributed to the recognition of clinically significant gas accumulations in live and dead cetaceans and pinnipeds. Understanding the appropriate application and limitations of the available imaging modalities is important for accurate interpretation of results. The presence of gas may be asymptomatic and must be interpreted cautiously alongside all other available data including clinical examination, clinical laboratory testing, gas analysis, necropsy examination, and histology results.
Schematic summary of different interactions between atrial fibrillation, oxidative stress, and flow abnormalities. Abbreviations should be included here are explained in the text.
Induction of ventricular-flow abnormalities in the ventricles during rapid atrial pacing (RAP) in comparison to unpaced controls using a porcine model. Effects of dronedarone and amiodarone demonstrated. Abbreviations are explained in the text adopted from Bukowska et al. (2012).
Effect of dronedarone on size of acute myocardial infarctions adopted from Qiu et al. (2006).
Patients with atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non-ST-segment elevation myocardial infarction) during an acute episode of AF. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography, which suggests that AF itself influences myocardial blood flow. The present review summarizes the effect of AF on the occurrence of ventricular oxidative stress, redox-sensitive signaling pathways and gene expression, and microcirculatory flow abnormalities in the left ventricle.
The G1/S transition is a crucial decision point in the cell cycle. At G1/S, there is an abrupt switch from a state of high cyclin-dependent kinases (CDK) inhibitor (CKI) levels and low S-phase CDK activity to a state of high S-phase CDK activity and degraded CKI. In budding yeast, this transition is triggered by phosphorylation of the Cdk1 inhibitor Sic1 at multiple sites by G1-phase CDK (Cln1,2-Cdk1) and S-phase CDK (Clb5,6-Cdk1) complexes. Using mathematical modeling we demonstrate that the mechanistic basis for the abruptness of the G1/S transition is the highly specific phosphorylation of Sic1 by S-phase CDK complex. This switch is generated by a double-negative feedback loop in which S-CDK1 phosphorylates Sic1, thus targeting it for destruction, and thereby liberating further S-CDK1 from the inhibitory Sic1-S-CDK1 complex. Our model predicts that the abruptness of the switch depends upon a strong binding affinity within the Sic1-S-CDK inhibitory complex. In vitro phosphorylation analysis using purified yeast proteins revealed that free Clb5-Cdk1 can create positive feedback by phosphorylating Sic1 that is bound in the inhibitory complex, and that Sic1 inhibits Clb5-Cdk1 with a sub-nanomolar inhibition constant. Our model also predicts that if the G1-phase CDK complex is too efficient at targeting Sic1 for destruction, then G1/S becomes a smooth and readily reversible transition. We propose that the optimal role for the G1-phase CDK in the switch would not be to act as a kinase activity directly responsible for abrupt degradation of CKI, but rather to act as a priming signal that initiates a positive feedback loop driven by emerging free S-phase CDK.
The anadromous salmonid life cycle includes both fresh water (FW) and seawater (SW) stages. The parr-smolt transformation (smoltification) pre-adapt the fish to SW while still in FW. The osmoregulatory organs change their mode of action from a role of preventing water inflow in FW, to absorb ions to replace water lost by osmosis in SW. During smoltification, the drinking rate increases, in the intestine the ion and fluid transport increases and is further elevated after SW entry. In SW, the intestine absorbs ions to create an inwardly directed water flow which is accomplished by increased Na(+), K(+)-ATPase (NKA) activity in the basolateral membrane, driving ion absorption via ion channels and/or co-transporters. This review will aim at discussing the expression patterns of the ion transporting proteins involved in intestinal fluid absorption in the FW stage, during smoltification and after SW entry. Of equal importance for intestinal fluid absorption as the active absorption of ions is the permeability of the epithelium to ions and water. During the smoltification the increase in NKA activity and water uptake in SW is accompanied by decreased paracellular permeability suggesting a redirection of the fluid movement from a paracellular route in FW, to a transcellular route in SW. Increased transcellular fluid absorption could be achieved by incorporation of aquaporins (AQPs) into the enterocyte membranes and/or by a change in fatty acid profile of the enterocyte lipid bilayer. An increased incorporation of unsaturated fatty acids into the membrane phospholipids will increase water permeability by enhancing the fluidity of the membrane. A second aim of the present review is therefore to discuss the presence and regulation of expression of AQPs in the enterocyte membrane as well as to discuss the profile of fatty acids present in the membrane phospholipids during different stages of the salmonid lifecycle.
Original recording of the lumen-positive transepithelial voltage (Vte) and voltage deflections (Δ V0) in an isolated, perfused mouse medullary thick ascending limb (mTAL). Steady state conditions were reached after about 15 min. The arrow indicates the time point used to quantify transport. In addition, the reversible blocking effect of luminal furosemide (100 μ M) is shown.
Basal transport values (Vte, Rte, and I'sc) of mouse mTALs plotted as a function of age (in days) of P2Y2 receptor WT (A, B, C; n = 30) and KO (D, E, F; n = 19) mouse. All 6 data panels include regression line fits where the stippled lines indicate the 95% confidence intervals.
Ion transport parameters in mTALs (A: Vte, B: Rte, and C: I'sc) from juvenile (25–35 days) and adult (35–55 days) P2Y2 receptor WT and KO mice (*statistical significance p < 0.05).
Western blot revealing similar expression of NKCC2 in P2Y2 receptor WT and KO (n = 3 each) inner stripe of outer medulla kidney tissue. Right side: The NKCC2 densitometric analysis is shown in reference to the equal dye loading control with β -actin.
AVP-stimulated NaCl transport increases in P2Y2 receptor WT and KO mTALs. (A) An original recording of the AVP-induced (10 nM) NaCl transport stimulation, in a P2Y2 receptor WT mouse mTAL. (B) AVP-stimulated NaCl transport was studied in P2Y2 receptor WT (n = 11) and KO mice mTALs (n = 6). Summary of this results is presented as transport changes (± I'sc) in NaCl transport.
Local purinergic signals modulate renal tubular transport. Acute activation of renal epithelial P2 receptors causes inhibition of epithelial transport and thus, should favor increased water and salt excretion by the kidney. So far only a few studies have addressed the effects of extracellular nucleotides on ion transport in the thick ascending limb. In the medullary thick ascending limb (mTAL), basolateral P2X receptors markedly (~25%) inhibit NaCl absorption. Although this segment does express both apical and basolateral P2Y2 receptors, acute activation of the basolateral P2Y2 receptors had no apparent effect on transepithelial ion transport. Here we studied, if the absence of the P2Y2 receptor causes chronic alterations in mTAL NaCl absorption by comparing basal and AVP-stimulated transepithelial transport rates. We used perfused mouse mTALs to electrically measure NaCl absorption in juvenile (35 days) male mice. Using microelectrodes, we determined the transepithelial voltage (Vte) and the transepithelial resistance (Rte) and thus, transepithelial NaCl absorption (equivalent short circuit current, I’sc). We find that mTALs from adult wild type (WT) mice have significantly lower NaCl absorption rates when compared to mTALs from juvenile WT mice. This could be attributed to significantly higher Rte values in mTALs from adult WT mice. This pattern was not observed in mTALs from P2Y2 receptor knockout (KO) mice. In addition, adult P2Y2 receptor KO mTALs have significantly lower Vte values compared to the juvenile. No difference in absolute I´sc was observed when comparing mTALs from WT and KO mice. AVP stimulated the mTALs to similar increases of NaCl absorption irrespective of the absence of the P2Y2 receptor. No difference was observed in the medullary expression level of NKCC2 in between the genotypes. These data indicate that the lack of P2Y2 receptors does not cause substantial differences in resting and AVP-stimulated NaCl absorption in
The role of aquaporin water channels in Elasmobanchs such as the dogfish Squalus acanthias is completely unknown. This investigation determines the expression and cellular and sub-cellular localization of AQP4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2). Western blots using the AQP4/1 antibody showed two bands (35.5kDa and 49.5kDa) in most tissues similar to mammals. Liver and rectal gland showed further bands. However, unlike in mammals, AQP4 protein was expressed in all tissues including respiratory tract and liver. The AQP4/2 antibody appeared much less specific in blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments. AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the cell including the nucleus. In rectal gland and cardiac stomach AQP4 was localized to secretary tubules but again AQP/1 and AQP/2 showed different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane and sometimes cytoplasmic distribution. Two types of large mitochondria-rich cells are known to exist in elasmobranches, that express either Na,K ATPase or V-type ATPase. Using Na,K-ATPase and V-type ATPase antibodies, AQP4 was colocalized with these proteins using the AQP4/1 antibody. Results show AQP4 is expressed in both (and all) branchial Na,K ATPase and V-type ATPase expressing cells.
The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5' and 3' RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (P(f)) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.
Top-cited authors
George Billman
  • The Ohio State University
Marco Vincenzo Narici
  • University of Padova
Billy Sperlich
  • University of Wuerzburg
Urs Granacher
  • University of Freiburg
William Mitchell
  • University of Nottingham