Inducible mouse model of skeletal muscle specific deletion of the Vitamin D Receptor (VDR)

Fil: Centeno, Viviana Andrea. Universidad Nacional de Córdoba. Facultad de Odontología. Cátedra Química Biológica A; Argentina.Fil: Sato, AY. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Fil: Cregor, M. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Fil: Akel, NS. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Fil: Bellido, T.. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Vitamin D3 has beneficial effects on skeletal muscle and can prevent falls leading to reduced bone fracture risk. Excess of glucocorticoids (GC), either endogenous as in aging or due to glucocorticoid administration as immunosuppressants, leads to muscle loss mass and increases the risk of bone fractures. Earlier findings showed that 1,25(OH)2 vitamin D3 (1,25D3) prevents GC-induced skeletal muscle atrophy in vivo, in muscle organ cultures ex vivo, and in C2C12 myoblasts/ myotubes in vitro. Based on these findings, we formulated the hypothesis that the beneficial actions of Vitamin D3 are mediated by direct hormonal effects on skeletal muscle cells. The purpose of this work was to generate mice lacking the receptor for Vitamin D (VDR) in skeletal muscle and test their response to Vitamin D3 signaling. Towards this end, we crossed transgenic mice expressing a tamoxifen-inducible Mer-Cre-Mer driven by Human skeletal muscle alfa actin promoter with mice whose Receptor for Vitamin D, VDR, was flanked with LoxP sites at the exon 3 locus of the gene. So, tamoxifen-induced specific activation of Cre, produces a new genomic structure of VDR. Males and females 3 months old mice VDRf/f;human skeletal muscle α-actin (HSA)-Cre+/- and their littermate control VDRf/f;HSA-Cre-/- mice (C) were injected with tamoxifen for 5 days (2mg/d 1x/d for 5d). In some experiments, vehicle was injected to check the effects of tamoxifen. Fifteen days after the last tamoxifen injection, at 4months old mice, animals were implanted with slow-release pellets of 2.1mg/kg/d prednisolone or placebo and were treated with 50ng/kg/d 1,25D3 or vehicle 5x/wk for 4wks. Mice were fed a regular Vitamin D3-containing diet and maintained in a 12h light/dark cycle. First, we confirmed tissue-specific VDR deletion. HSA-CRE was present in gDNA of CRE positive mice. Also, we confirmed the deletion of VDR only in induced by tamoxifen in CRE-positive mice. The excised form of the VDR is only detected in skeletal muscle (plantaris and tibialis anterior), but not in kidney, intestine, or bone, of Cre positive mice (VDR f/f;HSA-Cre +/-) treated with tamoxifen. VDR deletion induced by tamoxifen is only detected in CRE positive mice (VDR f/f;HSA-Cre +/-), but not in any tissues from control littermate mice (VDR fl/fl;HSA-Cre -/-). In conclusion this model achieves adult-onset deletion of the VDR in skeletal muscle (Cre and tamoxifen dependent) and thus it will allow its use to determine the direct effects of vitamin D3 signaling in this tissue.Fil: Centeno, Viviana Andrea. Universidad Nacional de Córdoba. Facultad de Odontología. Cátedra Química Biológica A; Argentina.Fil: Sato, AY. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Fil: Cregor, M. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Fil: Akel, NS. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Fil: Bellido, T.. Indiana University School of Medicine. Department of Anatomy & Cell Biology; United States.Bioquímica y Biología Molecular (ídem 1.6.3

Centimeter-long electron transport in marine sediments via conductive minerals

© 2015 International Society for Microbial Ecology All rights reserved. Centimeter-long electron conduction through marine sediments, in which electrons derived from sulfide in anoxic sediments are transported to oxygen in surficial sediments, may have an important influence on sediment geochemistry. Filamentous bacteria have been proposed to mediate the electron transport, but the filament conductivity could not be verified and other mechanisms are possible. Surprisingly, previous investigations have never actually measured the sediment conductivity or its basic physical properties. Here we report direct measurements that demonstrate centimeter-long electron flow through marine sediments, with conductivities sufficient to account for previously estimated electron fluxes. Conductivity was lost for oxidized sediments, which contrasts with the previously described increase in the conductivity of microbial biofilms upon oxidation. Adding pyrite to the sediments significantly enhanced the conductivity. These results suggest that the role of conductive minerals, which are more commonly found in sediments than centimeter-long microbial filaments, need to be considered when modeling marine sediment biogeochemistry

Fractional Cauchy problems on bounded domains: survey of recent results

In a fractional Cauchy problem, the usual first order time derivative is replaced by a fractional derivative. This problem was first considered by \citet{nigmatullin}, and \citet{zaslavsky} in $\mathbb R^d$ for modeling some physical phenomena. The fractional derivative models time delays in a diffusion process. We will give a survey of the recent results on the fractional Cauchy problem and its generalizations on bounded domains D\subset \rd obtained in \citet{m-n-v-aop, mnv-2}. We also study the solutions of fractional Cauchy problem where the first time derivative is replaced with an infinite sum of fractional derivatives. We point out a connection to eigenvalue problems for the fractional time operators considered. The solutions to the eigenvalue problems are expressed by Mittag-Leffler functions and its generalized versions. The stochastic solution of the eigenvalue problems for the fractional derivatives are given by inverse subordinators

Thermal and Sedimentation Stress Are Unlikely Causes of Brown Spot Syndrome in the Coral Reef Sponge, Ianthella basta

Background: Marine diseases are being increasingly linked to anthropogenic factors including global and local stressors. On the Great Barrier Reef, up to 66% of the Ianthella basta population was recently found to be afflicted by a syndrome characterized by brown spot lesions and necrotic tissue.\ud \ud Methodology/Principal Findings: Manipulative experiments were undertaken to ascertain the role of environmental stressors in this syndrome. Specifically, the effects of elevated temperature and sedimentation on sponge health and symbiont stability in I. basta were examined. Neither elevated temperature nor increased sedimentation were responsible for the brown spot lesions, but sponges exposed to 32°C developed substantial discoloration and deterioration of their tissues, resulting in death after eight days and a higher microbial diversity in those samples. No shifts in the microbial community of I. basta were observed across a latitudinal gradient or with increased sedimentation, with three previously described symbionts dominating the community of all sponges (Alphaproteobacteria, Gammaproteobacteria and Thaumarchaea).\ud \ud Conclusions/Significance: Results from this study highlight the stable microbial community of I. basta and indicate that thermal and sedimentation stress are not responsible for the brown spot lesions currently affecting this abundant and ecologically important sponge species

Modeling inflammation and oxidative stress in gastrointestinal disease development using novel organotypic culture systems

Gastroesophageal reflux disease (GERD), Barrett's esophagus (BE), graft-versus-host disease (GVHD), and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are common human gastrointestinal diseases that share inflammation as a key driver for their development. A general outcome resulting from these chronic inflammatory conditions is increased oxidative stress. Oxidative stress is caused by the generation of reactive oxygen and nitrogen species that are part of the normal inflammatory response, but are also capable of damaging cellular DNA, protein, and organelles. Damage to DNA can include DNA strand breaks, point mutations due to DNA adducts, as well as alterations in methylation patterns leading to activation of oncogenes or inactivation of tumor suppressors. There are a number of significant long-term consequences associated with chronic oxidative stress, most notably cancer. Infiltrating immune cells and stromal components of tissue including fibroblasts contribute to dynamic changes occurring in tissue related to disease development. Immune cells can potentiate oxidative stress, and fibroblasts have the capacity to contribute to advanced growth and proliferation of the epithelium and any resultant cancers. Disease models for GERD, BE, GVHD, and ulcerative colitis based on three-dimensional human cell and tissue culture systems that recapitulate in vivo growth and differentiation in inflammatory-associated microphysiological environments would enhance our understanding of disease progression and improve our ability to test for disease-prevention strategies. The development of physiologically relevant, human cell-based culture systems is therefore a major focus of our research. These novel models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders, and will have a translational impact allowing us to more rapidly develop therapeutic and chemopreventive agents. In summary, this work to develop advanced human cell-based models of inflammatory conditions will greatly improve our ability to study, prevent, and treat GERD, BE, GVHD, and inflammatory bowel disease. The work will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions

The what and where of adding channel noise to the Hodgkin-Huxley equations

One of the most celebrated successes in computational biology is the Hodgkin-Huxley framework for modeling electrically active cells. This framework, expressed through a set of differential equations, synthesizes the impact of ionic currents on a cell's voltage -- and the highly nonlinear impact of that voltage back on the currents themselves -- into the rapid push and pull of the action potential. Latter studies confirmed that these cellular dynamics are orchestrated by individual ion channels, whose conformational changes regulate the conductance of each ionic current. Thus, kinetic equations familiar from physical chemistry are the natural setting for describing conductances; for small-to-moderate numbers of channels, these will predict fluctuations in conductances and stochasticity in the resulting action potentials. At first glance, the kinetic equations provide a far more complex (and higher-dimensional) description than the original Hodgkin-Huxley equations. This has prompted more than a decade of efforts to capture channel fluctuations with noise terms added to the Hodgkin-Huxley equations. Many of these approaches, while intuitively appealing, produce quantitative errors when compared to kinetic equations; others, as only very recently demonstrated, are both accurate and relatively simple. We review what works, what doesn't, and why, seeking to build a bridge to well-established results for the deterministic Hodgkin-Huxley equations. As such, we hope that this review will speed emerging studies of how channel noise modulates electrophysiological dynamics and function. We supply user-friendly Matlab simulation code of these stochastic versions of the Hodgkin-Huxley equations on the ModelDB website (accession number 138950) and http://www.amath.washington.edu/~etsb/tutorials.html.Comment: 14 pages, 3 figures, review articl

Image-Based Assessment of Growth and Signaling Changes in Cancer Cells Mediated by Direct Cell-Cell Contact

Many important biological processes are controlled through cell-cell interactions, including the colonization of metastatic tumor cells and the control of differentiation of stem cells within their niche. Despite the crucial importance of the cellular environment in regulating cellular signaling, in vitro methods for the study of such interactions are difficult and/or indirect.We report on the development of an image-based method for distinguishing two cell types grown in coculture. Furthermore, cells of one type that are in direct contact with cells of a second type (adjacent cells) can be analyzed separately from cells that are not within a single well. Changes are evaluated using population statistics, which are useful in detecting subtle changes across two populations. We have used this system to characterize changes in the LNCaP prostate carcinoma cell line when grown in contact with human vascular endothelial cells (HUVECs). We find that the expression and phosphorylation of WWOX is reduced in LNCaP cells when grown in direct contact with HUVECs. Reduced WWOX signaling has been associated with reduced activation or expression of JNK and p73. We find that p73 levels are also reduced in LNCaP cells grown in contact with HUVECs, but we did not observe such a change in JNK levels.We find that the method described is statistically robust and can be adapted to a wide variety of studies where cell function or signaling are affected by heterotypic cell-cell contact. Ironically, a potential challenge to the method is its high level of sensitivity is capable of classifying events as statistically significant (due to the high number cells evaluated individually), when the biological effect may be less clear. The methodology would be best used in conjunction with additional methods to evaluate the biological role of potentially subtle differences between populations. However, many important events, such as the establishment of a metastatic tumor, occur through rare but important changes, and methods such as we describe here can be used to identify and characterize the contribution of the environment to these changes

Vitamin D and Systemic Lupus Erythematosus: Bones, Muscles, and Joints

Vitamin D3, or cholecalciferol, is the naturally occurring form of vitamin D that is converted in the skin and hydroxylated in the liver and kidney to the active form found in humans. The main role for vitamin D is calcium homeostasis, and low levels of vitamin D result in lower gastrointestinal absorption of calcium. Vitamin D is also critical for mineralization of bone tissue, muscle function, and coordination. Recent studies have found prevention of bone mass loss and reduction in falls and fractures in patients supplemented with vitamin D. A high percentage of systemic lupus erythematosus patients are reported to have insufficient or deficient levels of vitamin D. This paper reviews the biology of vitamin D, its role in calcium homeostasis, and how it contributes to the maintenance of bone, muscle, and joint function in older adults and individuals with systemic lupus erythematosus

Blood volume measurement with indocyanine green pulse spectrophotometry: dose and site of dye administration

(1) To determine the optimal administration site and dose of indocyanine green (ICG) for blood volume measurement using pulse spectrophotometry, (2) to assess the variation in repeated blood volume measurements for patients after subarachnoid hemorrhage and (3) to evaluate the safety and efficacy of this technique in patients who were treated for an intracranial aneurysm. Four repeated measurements of blood volume (BV) were performed in random order of bolus dose (10 mg or 25 mg ICG) and venous administration site (peripheral or central) in eight patients admitted for treatment of an intracranial aneurysm. Another five patients with subarachnoid hemorrhage underwent three repeated BV measurements with 25 mg ICG at the same administration site to assess the coefficient of variation. The mean +/- SD in BV was 4.38 +/- 0.88 l (n = 25) and 4.69 +/- 1.11 l (n = 26) for 10 mg and 25 mg ICG, respectively. The mean +/- SD in BV was 4.59 +/- 1.15 l (n = 26) and 4.48 +/- 0.86 l (n = 25) for central and peripheral administration, respectively. No significant difference was found. The coefficient of variance of BV measurement with 25 mg of ICG was 7.5% (95% CI: 3-12%). There is no significant difference between intravenous administration of either 10 or 25 mg ICG, and this can be injected through either a peripheral or central venous catheter. The 7.5% coefficient of variation in BV measurements determines the detectable differences using ICG pulse spectrophotometr

Microenvironment Changes (in pH) Affect VEGF Alternative Splicing

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl2 (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p < 0.001, when compared to VEGF145, 165 or 189). This was accompanied by activation of the p38 stress pathway and SR proteins (splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process