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Sample records for conditional double knockout

  1. Generation of conditional knockout mice.

    PubMed

    Sakamoto, Kazuhito; Gurumurthy, Channabasavaiah B; Wagner, Kay-Uwe

    2014-01-01

    Conditional knockout mouse models are powerful tools to examine the biological and molecular function(s) of genes in specific tissues. The general procedure to generate such genetically engineered mouse models consists of three main steps. The first step is to find the appropriate genomic clone of the gene of interest and to design the cloning and Southern blot strategies. The second step is the cloning of the gene-targeting vector with all its essential components including positive and negative selection cassettes and the insertion of LoxP sites. Although conventional methods are still being widely used for DNA cloning, we describe in this book chapter the use of λ Red phage-based homologous recombination in Escherichia coli to capture the genomic DNA of the gene of interest and to assemble the gene-targeting vector. This new method provides several advantages as it does not require the presence of restriction sites within the gene of interest to insert LoxP-flanked DNA fragments. In the final step, the gene-targeting vector is transferred into embryonic stem (ES) cells, and successfully targeted ES cell clones are injected into mouse blastocysts to generate conditional knockout mice. PMID:25064096

  2. Impaired conditioned taste aversion learning in spinophilin knockout mice.

    PubMed

    Stafstrom-Davis, C A; Ouimet, C C; Feng, J; Allen, P B; Greengard, P; Houpt, T A

    2001-01-01

    Plasticity in dendritic spines may underlie learning and memory. Spinophilin, a protein enriched in dendritic spines, has the properties of a scaffolding protein and is believed to regulate actin cytoskeletal dynamics affecting dendritic spine morphology. It also binds protein phosphatase-1 (PP-1), an enzyme that regulates dendritic spine physiology. In this study, we tested the role of spinophilin in conditioned taste aversion learning (CTA) using transgenic spinophilin knockout mice. CTA is a form of associative learning in which an animal rejects a food that has been paired previously with a toxic effect (e.g., a sucrose solution paired with a malaise-inducing injection of lithium chloride). Acquisition and extinction of CTA was tested in spinophilin knockout and wild-type mice using taste solutions (sucrose or sodium chloride) or flavors (Kool-Aid) paired with moderate or high doses of LiCl (0.15 M, 20 or 40 mL/kg). When sucrose or NaCl solutions were paired with a moderate dose of LiCl, spinophilin knockout mice were unable to learn a CTA. At the higher dose, knockout mice acquired a CTA but extinguished more rapidly than wild-type mice. A more salient flavor stimulus (taste plus odor) revealed similar CTA learning at both doses of LiCl in both knockouts and wild types. Sensory processing in the knockouts appeared normal because knockout mice and wild-type mice expressed identical unconditioned taste preferences in two-bottle tests, and identical lying-on-belly responses to acute LiCl. We conclude that spinophilin is a candidate molecule required for normal CTA learning. PMID:11584074

  3. Beyond knockouts: cre resources for conditional mutagenesis

    PubMed Central

    Murray, Stephen A.; Eppig, Janan T.; Smedley, Damian; Simpson, Elizabeth M.; Rosenthal, Nadia

    2013-01-01

    With the effort of the International Phenotyping Consortium (IMPC) to produce thousands of strains with conditional potential gathering steam, there is growing recognition that it must be supported by a rich toolbox of cre driver strains. The approaches to build cre strains have evolved in both sophistication and reliability, replacing first generation strains with tools that can target individual cell populations with incredible precision and specificity. The modest set of cre drivers generated by individual labs over the past 15+ years is now growing rapidly, thanks to a number of large-scale projects to produce new cre strains for the community. The power of this growing resource, however, depends upon the proper deep characterization of strain function, as even the best designed strain can display a variety of undesirable features that must be considered in experimental design. This must be coupled with the parallel development of informatics tools to provide functional data to the user, and facilitated access to the strains through public repositories. We will discuss the current progress on all of these fronts and the challenges that remain to ensure the scientific community can capitalize on the tremendous number of mouse resources at their disposal. PMID:22926223

  4. Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice

    PubMed Central

    Jeoung, Nam Ho; Rahimi, Yasmeen; Wu, Pengfei; Lee, W. N. Paul; Harris, Robert A.

    2015-01-01

    The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4. PMID:22360721

  5. Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

    SciTech Connect

    Yee, C M; Collette, N M; Loots, G G

    2011-07-29

    In the United States, {approx}5% are born with congenital birth defects due to abnormal function of cellular processes and interactions. Sclerosteosis, a rare autosomal recessive disease, causes hyperostosis of the axial and appendicular skeleton, and patients present radial deviation, digit syndactyly, nail dysplasia, and overall high bone mineral density. Sclerosteosis is due to a loss of function of sclerostin (Sost). Sost is a Wnt (abbrev.) antagonist; when mutated, nonfunctional Sost results in hyperactive osteoblast activity which leads to abnormal high bone mass. Previous studies have shown that Sost overexpression in transgenic mice causes reduced bone mineral density and a variety of limb phenotypes ranging from lost, fused, and split phalanges. Consistent with clinical manifestations of Sclerosteosis, Sost knockout mice exhibit increased generalized bone mineral density and syndactyly of the digits. Sostdc1 is a paralog of Sost that has also been described as an antagonist of Wnt signaling, in developing tooth buds. Unlike Sost knockouts, Sostdc1 null mice do not display any limb abnormalities. To determine if Sost and Sostdc1 have redundant functions during limb patterning, we examined Sost; Sostdc1 mice determined that they exhibit a novel preaxial polydactyly phenotype with a low penetrance. LacZ staining, skeletal preparations, and in situ hybridization experiments were used to help characterize this novel phenotype and understand how this phenotype develops. We find Sost and Sostdc1 to have complementary expression patterns during limb development, and the loss of their expression alters the transcription of several key limb regulators, such as Fgf8, Shh and Grem.

  6. Atherosclerosis, inflammation and lipoprotein glomerulopathy in kidneys of apoE-/-/LDL-/- double knockout mice

    PubMed Central

    2010-01-01

    Background The apoE-/-/LDL-/- double knockout mice are bearing considerable structural homology to human atherosclerosis. We hypothesized, that advanced lesion formation in the renal artery is associated with kidney alterations in these mice. Methods Kidneys from apoE-/-/LDL-/- double knockout mice at the age of 80 weeks (n = 6) and C57/BL control mice (n = 5) were infused with Microfil, harvested and scanned with micro-CT (12 μm cubic voxels) and Nano-CT (900 nm cubic voxels). We quantitated the total vascular volume using micro-CT. Number and cross-sectional area (μm2) of glomeruli were measured using histology. Results At the age of 80 weeks, the renal total vascular volume fraction decreased significantly (p < 0.001) compared to controls. Moreover, the renal artery showed advanced atherosclerotic lesions with adventitial Vasa vasorum neovascularization. Perivascular inflammation was present in kidneys of apoE-/-/LDL-/- double knockout mice, predominantly involved are plasma cells and leucocytes. Glomeruli cross-sectional area (9959 ± 1083 μm2) and number (24.8 ± 4.5) increased in apoE-/-/LDL-/- double knockout mice compared to controls (3533 ± 398 μm2; 17.6 ± 3, respectively), whereas 41% of the total number of glomeruli showed evidence for lipoprotein associated glomerulopathy (LPG). Moreover, immunohistochemistry demonstrated capillary aneurysms of the glomeruli filled with factor 8 containing emboli. Conclusion The reduced intra-renal total vascular volume is associated with systemic atherosclerosis and glomeruli alterations in the apoE-/-/LDL-/- double knockout mouse model. PMID:20727187

  7. A Conditional Knockout Mouse Line of the Oxytocin Receptor

    PubMed Central

    Lee, Heon-Jin; Caldwell, Heather K.; Macbeth, Abbe H.; Tolu, Selen G.; Young, W. Scott

    2008-01-01

    Oxytocin plays important roles in reproductive physiology and various behaviors, including maternal behavior and social memory. Its receptor (Oxtr) is present in peripheral tissues and brain, so a conditional knockout (KO, −/−) would be useful to allow elimination of the receptor in specific sites at defined times. We created a line of mice in which loxP sites flank Oxtr coding sequence (floxed) enable Cre recombinase-mediated inactivation of the receptor. We expressed Cre recombinase in these mice either in all tissues (Oxtr−/−) or the forebrain (OxtrFB/FB) using the Ca2+/calmodulin-dependent protein kinase IIα promoter. The latter KO has reduced Oxtr binding beginning 21–28 d postnatally, leading to prominent reductions in the lateral septum, hippocampus, and ventral pallidum. The medial amygdala is spared, and there is significant retention of binding within the olfactory bulb and nucleus and neocortex. We did not observe any deficits in the general health, sensorimotor functions, anxiety-like behaviors, or sucrose intake in either Oxtr−/− or OxtrFB/FB mice. Females of both KO types deliver pups, but only the OxtrFB/FB mice are able to eject milk. Oxtr−/− males show impaired social memory for familiar females, whereas the OxtrFB/FB males appear to recognize their species but not individuals. Our results confirm the importance of oxytocin in social recognition and demonstrate that spatial and temporal inactivation of the Oxtr will enable finer understanding of the physiological, behavioral, and developmental roles of the receptor. PMID:18356275

  8. Nitrotyrosinylation, Remodeling and Endothelial-Myocyte Uncoupling in iNOS, Cystathionine Beta Synthase (CBS) Knockouts and iNOS/CBS Double Knockout Mice

    PubMed Central

    Kundu, Soumi; Kumar, Munish; Sen, Utpal; Mishra, Paras K.; Tyagi, Neetu; Metreveli, Naira; Lominadze, David; Rodriguez, Walter; Tyagi, Suresh C.

    2009-01-01

    Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial-myocyte (E–M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy-mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS−/+/iNOS−/− double knockout, and wild-type (WT) mice. The levels of nitrotyrosine, MMP-2 and -9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial-myocyte function was determined in cardiac rings. In CBS−/+ mice, homocysteine was elevated and in iNOS−/− mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase-9 (MMP-9) levels were elevated in double knockout and CBS−/+ as compared to WT mice. Although MMP-2 levels were similar in CBS−/+, iNOS−/−, and CBS−/+/iNOS−/−, the levels were three- to fourfold higher than WT. The levels of collagen were similar in CBS−/+ and iNOS−/−, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS−/− and double knockout, and mildly increased in the CBS−/+, compared to WT mice. The endothelial-dependent relaxation was attenuated to the same extent in the CBS−/+ and iNOS−/−, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine

  9. Intragastric fat self-administration is impaired in GPR40/120 double knockout mice

    PubMed Central

    Sclafani, Anthony; Touzani, Khalid; Ackroff, Karen

    2015-01-01

    Mice acquire strong preferences for flavors paired with intragastric (IG) fat infusions. This IG fat conditioning is attenuated in double knockout (DoKO) mice missing GPR40 and GPR120 fatty acid receptors. Here we determined if GPR40/120 DoKO mice are also impaired in IG fat self-administration in an operant lick task. In daily 1-h sessions the mice were trained with a sipper spout that contained dry food pellets; licks on the spout triggered infusions of IG fat (Intralipid). The training sessions were followed by test sessions with an empty spout. GPR40/120 DoKO mice self-infused more 20% fat than wild type (WT) C57BL/6 mice in training with a food-baited spout (2.4 vs. 2.0 kcal/h) but self-infused less 20% fat than WT mice in empty spout tests (1.2 vs. 1.7 kcal/h). The DoKO mice also self-infused less 5% fat than WT mice (0.6 vs. 1.3 kcal/h) although both groups emitted more licks for 5% fat than 20% fat. The DoKO and WT mice did not differ, however, in their self-infusion of 12.5% glucose (1.5 vs. 1.6 kcal/h), which is isocaloric to 5% fat. A second 5% IL test showed that the DoKO mice reverted to a reduced self-infusion compared to WT mice. When the infusion was shifted to water, WT mice reduced licking in the first extinction session, whereas DoKO mice were less sensitive to the absence of infused fat. Our results indicate that post-oral GPR40/120 signaling is not required to process IG fat infusions in food-baited spout training sessions but contributes to post-oral fat reinforcement in empty spout tests and flavor conditioning tests. PMID:25911263

  10. Intragastric fat self-administration is impaired in GPR40/120 double knockout mice.

    PubMed

    Sclafani, Anthony; Touzani, Khalid; Ackroff, Karen

    2015-08-01

    Mice acquire strong preferences for flavors paired with intragastric (IG) fat infusions. This IG fat conditioning is attenuated in double knockout (DoKO) mice missing GPR40 and GPR120 fatty acid receptors. Here we determined if GPR40/120 DoKO mice are also impaired in IG fat self-administration in an operant lick task. In daily 1-h sessions the mice were trained with a sipper spout that contained dry food pellets; licks on the spout triggered infusions of IG fat (Intralipid). The training sessions were followed by test sessions with an empty spout. GPR40/120 DoKO mice self-infused more 20% fat than wild type (WT) C57BL/6 mice in training with a food-baited spout (2.4 vs. 2.0kcal/h) but self-infused less 20% fat than WT mice in empty spout tests (1.2 vs. 1.7kcal/h). The DoKO mice also self-infused less 5% fat than WT mice (0.6 vs. 1.3kcal/h) although both groups emitted more licks for 5% fat than 20% fat. The DoKO and WT mice did not differ, however, in their self-infusion of 12.5% glucose (1.5 vs. 1.6kcal/h), which is isocaloric to 5% fat. A second 5% IL test showed that the DoKO mice reverted to a reduced self-infusion compared to WT mice. When the infusion was shifted to water, WT mice reduced licking in the first extinction session, whereas DoKO mice were less sensitive to the absence of infused fat. Our results indicate that post-oral GPR40/120 signaling is not required to process IG fat infusions in food-baited spout training sessions but contributes to post-oral fat reinforcement in empty spout tests and flavor conditioning tests. PMID:25911263

  11. Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

    PubMed

    Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

    2014-01-01

    Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy. PMID:25064116

  12. Normal Maternal Behavior, But Increased Pup Mortality, in Conditional Oxytocin Receptor Knockout Females

    PubMed Central

    Macbeth, Abbe H.; Stepp, Jennifer E.; Lee, Heon-Jin; Young, W. Scott; Caldwell, Heather K.

    2011-01-01

    Oxytocin (Oxt) and the Oxt receptor (Oxtr) are implicated in the onset of maternal behavior in a variety of species. Recently, we developed two Oxtr knockout lines: a total body knockout (Oxtr−/−) and a conditional Oxtr knockout (OxtrFB/FB) in which the Oxtr is lacking only in regions of the forebrain, allowing knockout females to potentially nurse and care for their biological offspring. In the current study, we assessed maternal behavior of postpartum OxtrFB/FB females toward their own pups and maternal behavior of virgin Oxtr−/− females toward foster pups and compared knockouts of both lines to wildtype (Oxtr+/+) littermates. We found that both Oxtr−/− and OxtrFB/FB females appear to have largely normal maternal behaviors. However, with first litters, approximately 40% of the OxtrFB/FB knockout dams experienced high pup mortality, compared to fewer than 10% of the Oxtr+/+ dams. We then went on to test whether or not this phenotype occurred in subsequent litters or when the dams were exposed to an environmental disturbance. We found that regardless of the degree of external disturbance, OxtrFB/FB females lost more pups on their first and second litters compared to wildtype females. Possible reasons for higher pup mortality in OxtrFB/FB females are discussed. PMID:20939667

  13. Exacerbated neuronal ceroid lipofuscinosis phenotype in Cln1/5 double-knockout mice

    PubMed Central

    Blom, Tea; Schmiedt, Mia-Lisa; Wong, Andrew M.; Kyttälä, Aija; Soronen, Jarkko; Jauhiainen, Matti; Tyynelä, Jaana; Cooper, Jonathan D.; Jalanko, Anu

    2013-01-01

    SUMMARY Both CLN1 and CLN5 deficiencies lead to severe neurodegenerative diseases of childhood, known as neuronal ceroid lipofuscinoses (NCLs). The broadly similar phenotypes of NCL mouse models, and the potential for interactions between NCL proteins, raise the possibility of shared or convergent disease mechanisms. To begin addressing these issues, we have developed a new mouse model lacking both Cln1 and Cln5 genes. These double-knockout (Cln1/5 dko) mice were fertile, showing a slight decrease in expected Mendelian breeding ratios, as well as impaired embryoid body formation by induced pluripotent stem cells derived from Cln1/5 dko fibroblasts. Typical disease manifestations of the NCLs, i.e. seizures and motor dysfunction, were detected at the age of 3 months, earlier than in either single knockout mouse. Pathological analyses revealed a similar exacerbation and earlier onset of disease in Cln1/5 dko mice, which exhibited a pronounced accumulation of autofluorescent storage material. Cortical demyelination and more pronounced glial activation in cortical and thalamic regions was followed by cortical neuron loss. Alterations in lipid metabolism in Cln1/5 dko showed a specific increase in plasma phospholipid transfer protein (PLTP) activity. Finally, gene expression profiling of Cln1/5 dko cortex revealed defects in myelination and immune response pathways, with a prominent downregulation of α-synuclein in Cln1/5 dko mouse brains. The simultaneous loss of both Cln1 and Cln5 genes might enhance the typical pathological phenotypes of these mice by disrupting or downregulating shared or convergent pathogenic pathways, which could potentially include interactions of CLN1 and CLN5. PMID:23065637

  14. Molecular signatures of neurodegeneration in the cortex of PS1/PS2 double knockout mice

    PubMed Central

    Mirnics, Károly; Norstrom, Eric M; Garbett, Krassimira; Choi, Se Hoon; Zhang, Xiaoqiong; Ebert, Philip; Sisodia, Sangram S

    2008-01-01

    Background Familial Alzheimer's disease-linked variants of presenilin (PSEN1 and PSEN2) contribute to the pathophysiology of disease by both gain-of-function and loss-of-function mechanisms. Deletions of PSEN1 and PSEN2 in the mouse forebrain result in a strong and progressive neurodegenerative phenotype which is characterized by both anatomical and behavioral changes. Results To better understand the molecular changes associated with these morphological and behavioral phenotypes, we performed a DNA microarray transcriptome profiling of the hippocampus and the frontal cortex of the PSEN1/PSEN2 double knock-out mice and littermate controls at five different ages ranging from 2–8 months. Our data suggest that combined deficiencies of PSEN1 and PSEN2 results in a progressive, age-dependent transcriptome signature related to neurodegeneration and neuroinflammation. While these events may progress differently in the hippocampus and frontal cortex, the most critical expression signatures are common across the two brain regions, and involve a strong upregulation of cathepsin and complement system transcripts. Conclusion The observed neuroinflammatory expression changes are likely to be causally linked to the neurodegenerative phenotype observed in mice with compound deletions of PSEN1 and PSEN2. Furthermore, our results suggest that the evaluation of inhibitors of PS/γ-secretase activity for treatment of Alzheimer's Disease must include close monitoring for signs of calpain-cathepsin system activation. PMID:18834536

  15. Impaired motor coordination and disrupted cerebellar architecture in Fgfr1 and Fgfr2 double knockout mice

    PubMed Central

    Smith, Karen Müller; Williamson, Theresa L.; Schwartz, Michael L.

    2012-01-01

    Fibroblast growth factor receptor (FGFR) signaling determines the size of the cerebral cortex by regulating the amplification of radial glial stem cells, and participates in the formation of midline glial structures. We show that Fgfr1 and Fgfr2 double knockouts (FGFR DKO) generated by Cre mediated recombination driven by the human GFAP promoter (hGFAP) have reduced cerebellar size due to reduced proliferation of radial glia and other glial precursors in late embryonic and neonatal FGFR DKO mice. The proliferation of granule cell progenitors (GCPs) in the EGL was also reduced, leading to reduced granule cell numbers. Furthermore, both inward migration of granule cells into the inner granule cell layer (IGL) and outward migration of GABA interneurons into the molecular layer (ML) were arrested, disrupting layer and lobular morphology. Purkinje neurons and their dendrites, which were not targeted by Cre mediated recombination of Fgf receptors, were also misplaced in FGFR DKO mice, possibly as a consequence of altered Bergmann glia orientation or reduced granule cell number. Our findings indicate a dual role for FGFR signaling in cerebellar morphogenesis. The first role is to amplify the number of granule neuron precursors in the external granular layer and glial precursor cells throughout the cerebellum. The second is to establish the correct Bergmann glia morphology, which is crucial for granule cell migration. The disrupted cerebellar size and laminar architecture resulting from loss of FGFR signaling impairs motor learning and coordination in FGFR DKO mice. PMID:22578469

  16. Directionality Effect in Double Conditionals.

    PubMed

    Espino, Orlando; Sánchez-Curbelo, Isana; Bolaños-Medina, Alicia

    2015-01-01

    Directionality effect in deductive reasoning is a very well-known phenomenon that shows that the percentage of forward or backward inferences that participants make depends on the conditional form used. A new extension of the semantic hypothesis (Oberauer & Wilhelm, 2000) is presented to explain the directionality effect in double conditionals with different directionality. This hypothesis claims that the directional effect depends on which term plays the role of relatum. It also makes several novel claims which have been confirmed in three experiments: Experiments 1 and 2 showed there were more forward than backward inferences when the end-term that played the role of relatum was in the first premise, experiment 1: t (45) = 2.73, p < .01, experiment 2: t (38) = 12.06, p < .05, but there were more backward than forward inferences when the end-term that played the role of relatum was in the second premise, experiment 1: t (45) = 2.84, p < .01, experiment 2: t (38) = 2.21, p < .04. Experiment 3 showed that there was no directional effect when both end-terms played the role of relatum, t (34) = 1.39, p = .17, or when both middle-terms (or neither of the end-terms) played the role of relatum, t (34) = .78, p = .44. These experiments confirmed the predictions of the new extension of the semantic hypothesis. PMID:26239471

  17. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.

    PubMed

    Shen, Zhongfu; Zhang, Xianliang; Chai, Yongping; Zhu, Zhiwen; Yi, Peishan; Feng, Guoxin; Li, Wei; Ou, Guangshuo

    2014-09-01

    Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research. PMID:25155554

  18. Perseverative instrumental and Pavlovian responding to conditioned stimuli in serotonin transporter knockout rats.

    PubMed

    Nonkes, Lourens J P; Homberg, Judith R

    2013-02-01

    Environmental stimuli can influence behavior via the process of Pavlovian conditioning. Recent genetic research suggests that some individuals are more sensitive to environmental stimuli for behavioral guidance than others. One important mediator of this effect is serotonin transporter (5-HTT) genetic variance, which increases sensitivity to Pavlovian conditioned stimuli through changes in the build-up of corticolimbic circuits. As these stimuli can have reinforcing effects on instrumental responding, we here investigated their effects on instrumental behavior in 5-HTT knockout rats and their wild-type counterparts by means of the signal attenuation paradigm. In this paradigm animals acquired a Pavlovian association between a stimulus and food reward, and subsequently they had to lever press in order to gain access to this food reward-associated stimulus. Thereafter, half of the animals underwent extinction training during which extinction of the primary Pavlovian association was induced via non-reinforced stimulus presentations, whereas the other half did not receive this training. During a final test session all animals were tested for instrumental responding for the non-reinforced Pavlovian conditioned stimulus, as well as instrumental and Pavlovian responding to the stimulus after an initial lever-press. No genotype differences were observed during the training and extinction sessions. However, during the test session 5-HTT knockout rats that had not received prior extinction training displayed excessive instrumental responding. This was specifically observed during presentation of the stimulus (induced by the first lever press) and was accompanied by an increased number of feeder visits after termination of the stimulus presentation. An additionally performed c-Fos immunohistochemistry study revealed that the behaviors in these animals were associated with abnormal c-Fos immunoreactivity in the orbitofrontal cortex and basolateral amygdala, regions important

  19. Sub-optimal phenotypes of double-knockout mutants of Escherichia coli depend on the order of gene deletions.

    PubMed

    Gawand, Pratish; Said Abukar, Fatumina; Venayak, Naveen; Partow, Siavash; Motter, Adilson E; Mahadevan, Radhakrishnan

    2015-08-01

    Metabolic networks are characterized by multiple redundant reactions that do not have a clear biological function. The redundancies in the metabolic networks are implicated in adaptation to random mutations and survival under different environmental conditions. Reactions that are not active under wild-type growth conditions, but get transiently activated after a mutation event such as gene deletion are known as latent reactions. Characterization of multiple-gene knockout mutants can identify the physiological roles of latent reactions. In this study, we characterized double-gene deletion mutants of E. coli with the aim of investigating the sub-optimal physiology of the mutants and the possible roles of latent reactions. Specifically, we investigated the effects of the deletion of the glyoxylate-shunt gene aceA (encoding a latent reaction enzyme, isocitrate lyase) on the growth characteristics of the mutant E. coli Δpgi. The deletion of aceA reduced the growth rate of E. coli Δpgi, indicating that the activation of the glyoxylate shunt plays an important role in adaptation of the mutant E. coli Δpgi when no other latent reactions are concurrently inactivated. We also investigated the effect of the order of the gene deletions on the growth rates and substrate uptake rates of the double-gene deletion mutants. The results indicate that the order in which genes are deleted determines the phenotype of the mutants during the sub-optimal growth phase. To elucidate the mechanism behind the difference between the observed phenotypes, we carried out transcriptomic analysis and constraint-based modeling of the mutants. Transcriptomic analysis showed differential expression of the gene aceK (encoding the protein isocitrate dehydrogenase kinase) involved in controlling the isocitrate flux through the TCA cycle and the glyoxylate shunt. Higher acetate production in the E. coli ΔaceA1 Δpgi2 mutant was consistent with the increased aceK expression, which limits the TCA cycle

  20. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs

    PubMed Central

    Liu, Ming; Wang, Xiu-Ping

    2015-01-01

    To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015]. PMID:26484260

  1. Conditional knockout of retinal determination genes in differentiating cells in Drosophila.

    PubMed

    Jin, Meng; Eblimit, Aiden; Pulikkathara, Merlyn; Corr, Stuart; Chen, Rui; Mardon, Graeme

    2016-08-01

    Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults. PMID:27257739

  2. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

    PubMed

    Jiang, Nan-Wei; Wang, De-Juan; Xie, Ya-Jun; Zhou, Liang; Su, Li-Da; Li, Huashun; Wang, Qin-Wen; Shen, Ying

    2016-06-01

    Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells. PMID:26194056

  3. Generation of a conditional knockout allele for the NFAT5 gene in mice.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity enhancer element binding protein (TonEBP) plays a crucial role in protection of renal medullary cells against hyperosmotic stress, urinary concentration, the adaptive immune response, and other physiological systems. Since it is also important for development, conventional homozygous-null mutations result in perinatal death, which hinders the analysis of NFAT5 function in specific tissues in vivo. Here we describe the generation of mice with a conditional-null allele, in which loxP sites are inserted around exon 4. Mice harboring the floxed allele (NFAT5(flx) ) were mated to a strain expressing a tamoxifen-inducible derivative of the Cre-recombinase (Cre (+)) under the control of the ubiqitinC promoter. The resultant homozygous conditional knockout mice (Cre (+) NFAT5 (flx/flx) ) are viable, fertile, and show normal expression of NFAT5 and NFAT5 target genes, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen in 8-week-old mice resulted in a decrease in NFAT5 expression of about 70-90% in all tested tissues (renal cortex, renal outer medulla, renal inner medulla, heart, lung, spleen, skeletal muscle). Accordingly, the expression of the NFAT5 target genes aldose reductase and heat shock protein 70 in the renal medulla was also significantly decreased. Mice harboring this conditional knockout allele should be useful in future studies for gaining a better understanding of tissue and cell-type specific functions of NFAT5 in adult animals under physiological and pathophysiological conditions. PMID:25601839

  4. Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Tushima, Anmi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Kuhara, Tomiko; Ohse, Morimasa; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Kobayashi, Keiko

    2011-12-01

    The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency. PMID:21908222

  5. The effects of cannabinoids on contextual conditioned fear in CB1 knockout and CD1 mice.

    PubMed

    Mikics, Eva; Dombi, Timea; Barsvári, Beáta; Varga, Balázs; Ledent, Catherine; Freund, Tamás F; Haller, József

    2006-05-01

    We studied the effects of cannabinoids on contextual conditioned fear responses. CB1 knockout and wild-type (CD1) mice were exposed to a brief session of electric shocks, and their behavior was studied in the same context 24 h later. In wild-type mice, shock exposure increased freezing and resting, and decreased locomotion and exploration. The genetic disruption of the CB1 receptor abolished the conditioned fear response. The CB1 antagonist AM-251 reduced the peak of the conditioned fear response when applied 30 min before behavioral testing (i.e. 24 h after shocks) in CD1 (wild-type) mice. The cannabinoid agonist WIN-55,212-2 markedly increased the conditioned fear response in CD1 mice, the effect of which was potently antagonized by AM-251. Thus, cannabinoid receptor activation appears to strongly promote the expression of contextual conditioned fear. In earlier experiments, cannabinoids did not interfere with the expression of cue-induced conditioned fear but strongly promoted its extinction. Considering the primordial role of the amygdala in simple associative learning (e.g. in cue-induced fear) and the role of the hippocampus in learning more complex stimulus relationships (e.g. in contextual fear), the present and earlier findings are not necessarily contradictory, but suggest that cannabinoid signaling plays different roles in the two structures. Data are interpreted in terms of the potential involvement of cannabinoids in trauma-induced behavioral changes. PMID:16572000

  6. Does murine spermatogenesis require WNT signalling? A lesson from Gpr177 conditional knockout mouse models.

    PubMed

    Chen, Su-Ren; Tang, J-X; Cheng, J-M; Hao, X-X; Wang, Y-Q; Wang, X-X; Liu, Y-X

    2016-01-01

    Wingless-related MMTV integration site (WNT) proteins and several other components of the WNT signalling pathway are expressed in the murine testes. However, mice mutant for WNT signalling effector β-catenin using different Cre drivers have phenotypes that are inconsistent with each other. The complexity and overlapping expression of WNT signalling cascades have prevented researchers from dissecting their function in spermatogenesis. Depletion of the Gpr177 gene (the mouse orthologue of Drosophila Wntless), which is required for the secretion of various WNTs, makes it possible to genetically dissect the overall effect of WNTs in testis development. In this study, the Gpr177 gene was conditionally depleted in germ cells (Gpr177(flox/flox), Mvh-Cre; Gpr177(flox/flox), Stra8-Cre) and Sertoli cells (Gpr177(flox/flox), Amh-Cre). No obvious defects in fertility and spermatogenesis were observed in these three Gpr177 conditional knockout (cKO) mice at 8 weeks. However, late-onset testicular atrophy and fertility decline in two germ cell-specific Gpr177 deletion mice were noted at 8 months. In contrast, we did not observe any abnormalities of spermatogenesis and fertility, even in 8-month-old Gpr177(flox/flox), Amh-Cre mice. Elevation of reactive oxygen species (ROS) was detected in Gpr177 cKO germ cells and Sertoli cells and exhibited an age-dependent manner. However, significant increase in the activity of Caspase 3 was only observed in germ cells from 8-month-old germ cell-specific Gpr177 knockout mice. In conclusion, GPR177 in Sertoli cells had no apparent influence on spermatogenesis, whereas loss of GPR177 in germ cells disrupted spermatogenesis in an age-dependent manner via elevating ROS levels and triggering germ cell apoptosis. PMID:27362799

  7. Conditional N-WASP knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology.

    PubMed

    Jain, Neeraj; Lim, Lee Wei; Tan, Wei Ting; George, Bhawana; Makeyev, Eugene; Thanabalu, Thirumaran

    2014-04-01

    Cerebrospinal fluid (CSF) is produced by the choroid plexus and moved by multi-ciliated ependymal cells through the ventricular system of the vertebrate brain. Defects in the ependymal layer functionality are a common cause of hydrocephalus. N-WASP (Neural-Wiskott Aldrich Syndrome Protein) is a brain-enriched regulator of actin cytoskeleton and N-WASP knockout caused embryonic lethality in mice with neural tube and cardiac abnormalities. To shed light on the role of N-WASP in mouse brain development, we generated N-WASP conditional knockout mouse model N-WASP(fl/fl); Nestin-Cre (NKO-Nes). NKO-Nes mice were born with Mendelian ratios but exhibited reduced growth characteristics compared to their littermates containing functional N-WASP alleles. Importantly, all NKO-Nes mice developed cranial deformities due to excessive CSF accumulation and did not survive past weaning. Coronal brain sections of these animals revealed dilated lateral ventricles, defects in ciliogenesis, loss of ependymal layer integrity, reduced thickness of cerebral cortex and aqueductal stenosis. Immunostaining for N-cadherin suggests that ependymal integrity in NKO-Nes mice is lost as compared to normal morphology in the wild-type controls. Moreover, scanning electron microscopy and immunofluorescence analyses of coronal brain sections with anti-acetylated tubulin antibodies revealed the absence of cilia in ventricular walls of NKO-Nes mice indicative of ciliogenesis defects. N-WASP deficiency does not lead to altered expression of N-WASP regulatory proteins, Fyn and Cdc42, which have been previously implicated in hydrocephalus pathology. Taken together, our results suggest that N-WASP plays a critical role in normal brain development and implicate actin cytoskeleton regulation as a vulnerable axis frequently deregulated in hydrocephalus. PMID:24462670

  8. A conditional knockout resource for the genome–wide study of mouse gene function

    PubMed Central

    Skarnes, William C.; Rosen, Barry; West, Anthony P.; Koutsourakis, Manousos; Bushell, Wendy; Iyer, Vivek; Mujica, Alejandro O.; Thomas, Mark; Harrow, Jennifer; Cox, Tony; Jackson, David; Severin, Jessica; Biggs, Patrick; Fu, Jun; Nefedov, Michael; de Jong, Pieter J.; Stewart, A. Francis; Bradley, Allan

    2013-01-01

    Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome. PMID:21677750

  9. Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

    PubMed

    Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

    2015-01-01

    Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

  10. Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

    PubMed Central

    MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

    2015-01-01

    Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

  11. Tissue- and cell-specific functions of the androgen receptor revealed through conditional knockout models in mice.

    PubMed

    De Gendt, Karel; Verhoeven, Guido

    2012-04-16

    This review aims to evaluate the contribution of individual cell-selective knockout models to our current understanding of androgen action. Cre/loxP technology has allowed the generation of cell-selective knockout models targeting the androgen receptor (AR) in distinct putative target cells in a wide variety of organs and tissues including: testis, ovary, accessory sex tissues, muscle, bone, fat, liver, skin and myeloid tissue. In some androgen-regulated processes such as spermatogenesis and folliculogenesis this approach has lead to the identification of a key cellular mediator of androgen action (Sertoli and granulosa cells, respectively). In many target tissues, however, the final response to androgens appears to be more complex. Here, cell-selective knockout technology offers a platform upon which we can begin to unravel the more complex interplay and signaling pathways of androgens. A prototypic example is the analysis of mesenchymal-epithelial interactions in many accessory sex glands. Furthermore, for some actions of testosterone, in which part of the effect is mediated by the active metabolite 17β-estradiol, conditional knockout technology offers a novel strategy to study the relative contribution of AR and estrogen receptor-mediated signaling. The latter approach has already resulted in a better understanding of androgen action in brain and bone. Finally, cell-selective knockout technology has generated valuable models to search for AR-controlled molecular mediators of androgen action, a strategy that has successfully been applied to the study of androgen action in the testis and in the epididymis. Although some conditional knockout models have provided clear answers to physiologic questions, it should be noted that others have pointed to unexpected complexities or technical limitations confounding interpretation of the results. PMID:21871526

  12. Generation and characterization of a mouse model of the metabolic syndrome: apolipoprotein E and aromatase double knockout mice.

    PubMed

    Scott, Nicola J A; Cameron, Vicky A; Raudsepp, Sara; Lewis, Lynley K; Simpson, Evan R; Richards, A Mark; Ellmers, Leigh J

    2012-03-01

    The aim of this study was to create a comprehensive mouse model of the metabolic syndrome by crossing aromatase-deficient (ArKO) mice with apolipoprotein E-deficient (ApoE(-/-)) mice. Successive crossbreeding of ArKO with ApoE(-/-)-deficient mice generated double knockout, MetS-Tg mice. The phenotypic characteristics of the MetS-Tg mice were assessed at 3, 6, and 12 mo of age and compared with age- and sex-matched wild-type (WT) controls. Blood pressure and heart rate were recorded by a noninvasive, computerized tail-cuff system. Oral glucose and intraperitoneal insulin tolerance tests were performed. Serum cholesterol levels were measured by a combined quantitative colorimetric assay. Plasma adiponectin, C-reactive protein (CRP), insulin, interleukin-6 (IL-6), leptin, resistin, and tumor necrosis factor-α (TNF-α) were measured by multiplexed ELISA. MetS-Tg mice displayed significantly increased body weight, central obesity, and elevated blood pressure at all three ages compared with WT mice. Elevated serum cholesterol was associated with higher triglycerides and LDL/VLDL cholesterol particles and was accompanied by a decrease in HDL and histological evidence of fatty liver. MetS-Tg mice of all ages showed impaired glucose tolerance. At 12 mo, MetS-Tg mice had elevated plasma levels of CRP, IL-6, leptin, and TNF-α, but resistin levels were largely unchanged. We now report that this combination of gene knockouts produces a novel strain of mice that display the diverse clinical features of the metabolic syndrome, including central obesity, progressive hypertension, an adverse serum lipid profile, fatty liver, glucose intolerance, insulin resistance, and evidence of an inflammatory state. PMID:22185842

  13. Conditional Knockout of Telomerase Reverse Transcriptase in Mesenchymal Cells Impairs Mouse Pulmonary Fibrosis

    PubMed Central

    Liu, Tianju; Yu, Hongfeng; Ding, Lin; Wu, Zhe; Gonzalez De Los Santos, Francina; Liu, Jianhua; Ullenbruch, Matthew; Hu, Biao; Martins, Vanessa; Phan, Sem H.

    2015-01-01

    Telomerase is typically expressed in cellular populations capable of extended replication, such as germ cells, tumor cells, and stem cells, but is also induced in tissue injury, repair and fibrosis. Its catalytic component, telomerase reverse transcriptase (TERT) is induced in lung fibroblasts from patients with fibrotic interstitial lung disease and in rodents with bleomycin-induced pulmonary fibrosis. To evaluate the fibroblast specific role of TERT in pulmonary fibrosis, transgenic mice bearing a floxed TERT allele were generated, and then crossed with an inducible collagen α2(I)-Cre mouse line to generate fibroblast specific TERT conditional knockout mice. TERT-specific deficiency in mesenchymal cells caused attenuation of pulmonary fibrosis as manifested by reduced lung hydroxyproline content, type I collagen and α-smooth muscle actin mRNA levels. The TERT-deficient mouse lung fibroblasts displayed decreased cell proliferative capacity and higher susceptibility to induced apoptosis compared with control cells. Additionally TERT deficiency was associated with heightened α-smooth muscle actin expression indicative of myofibroblast differentiation. However the impairment of cell proliferation and increased susceptibility to apoptosis would cause a reduction in the myofibroblast progenitor population necessary to mount a successful myofibroblast-dependent fibrotic response. These findings identified a key role for TERT in fibroblast proliferation and survival essential for pulmonary fibrosis. PMID:26555817

  14. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons.

    PubMed

    Jiang, Haisong; Kang, Sung-Ung; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Lee, Yong-Kyu; Kang, Bong-Gu; Lee, Yunjong; Zhang, Jianmin; Pletnikova, Olga; Troncoso, Juan C; Pirooznia, Shelia; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M

    2016-01-01

    Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function. PMID:27622213

  15. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption

    PubMed Central

    Ghosal, Abhisek; Lambrecht, Nils; Subramanya, Sandeep B.; Kapadia, Rubina

    2013-01-01

    The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health. PMID:23104561

  16. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons

    PubMed Central

    Jiang, Haisong; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Pletnikova, Olga; Troncoso, Juan C.; Pirooznia, Shelia; Andrabi, Shaida A.

    2016-01-01

    Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function. PMID:27622213

  17. Conditional knockout of pik3c3 causes a murine muscular dystrophy.

    PubMed

    Reifler, Aaron; Li, Xingli; Archambeau, Ashley J; McDade, Joel R; Sabha, Nesrin; Michele, Daniel E; Dowling, James J

    2014-06-01

    Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy. PMID:24726497

  18. Functional interplay between cylindromatosis and histone deacetylase 6 in ciliary homeostasis revealed by phenotypic analysis of double knockout mice.

    PubMed

    Ran, Jie; Yu, Fan; Qin, Juan; Zhang, Yijun; Yang, Yunfan; Li, Dengwen; Zhou, Jun; Liu, Min

    2016-05-10

    Cilia are present in most vertebrate tissues with a wide variety of functions, and abnormalities of cilia are linked to numerous human disorders. However, the molecular events underlying ciliary homeostasis are poorly understood. In this study, we generated double knockout (DKO) mice for the deubiquitinase cylindromatosis (CYLD) and histone deacetylase 6 (HDAC6), two critical ciliary regulators. The Cyld/Hdac6 DKO mice were phenotypically normal and showed no obvious variances in weight or behavior compared with their wild-type littermates. Strikingly, Cyld loss-induced ciliary defects in the testis, trachea, and kidney were abrogated in the Cyld/Hdac6 DKO mice. In addition, the diminished α-tubulin acetylation and impaired sonic hedgehog signaling caused by loss of Cyld were largely restored by simultaneous deletion of Hdac6. We further found by immunofluorescence microscopy a colocalization of CYLD and HDAC6 at the centrosome/basal body and, interestingly, loss of Cyld promoted the localization of HDAC6 at the centrosome/basal body. These findings provide physiological insight into the ciliary role of the CYLD/HDAC6 axis and suggest a functional interplay between these two proteins in ciliary homeostasis. PMID:27028867

  19. Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis.

    PubMed

    Kondo, Yoshitaka; Masutomi, Hirofumi; Noda, Yoshihiro; Ozawa, Yusuke; Takahashi, Keita; Handa, Setsuko; Maruyama, Naoki; Shimizu, Takahiko; Ishigami, Akihito

    2014-01-01

    Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca(2+) efflux by activating the calmodulin-dependent Ca(2+)-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15-24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers - ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion. PMID:25003023

  20. CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APC(Min)/CD24 double knockout transgenic mice.

    PubMed

    Naumov, Inna; Zilberberg, Alona; Shapira, Shiran; Avivi, Doran; Kazanov, Dina; Rosin-Arbesfeld, Rina; Arber, Nadir; Kraus, Sarah

    2014-09-01

    Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APC(Min/+) mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation-associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by β-catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis. PMID:24500912

  1. The hGFAP-driven conditional TSPO knockout is protective in a mouse model of multiple sclerosis

    PubMed Central

    Daugherty, Daniel J.; Chechneva, Olga; Mayrhofer, Florian; Deng, Wenbin

    2016-01-01

    The mitochondrial translocator protein (TSPO) has been implicated in CNS diseases. Here, we sought to determine the specific role of TSPO in experimental autoimmune encephalomyelitis (EAE), the most studied animal model of multiple sclerosis (MS). To fundamentally elucidate the functions of TSPO, we first developed a viable TSPO knockout mouse. A conditional TSPO knockout mouse was generated by utilizing the Cre-Lox system. We generated a TSPO floxed mouse, and then crossed this mouse with a Cre recombinase expressing mouse driven by the human glial fibrillary acidic protein (hGFAP) promoter. The resultant mouse was a neural linage line specific TSPO knockout. The loss of TSPO in the CNS did not result in overt developmental defects or phenotypes. The TSPO−/− mouse showed a decrease in GFAP expression, correlating with a decrease in astrogliosis in response to neural injury during EAE. This decrease in astrogliosis was also witnessed in the lessening of severity of EAE clinical scoring, indicating an in vivo functional role for TSPO in suppressing EAE. The TSPO−/− mouse could be a useful tool in better understanding the role of TSPO in CNS disease, and our results implicate TSPO as a potential therapeutic target in MS. PMID:26925573

  2. Induction of Fatal Inflammation in LDL Receptor and ApoA-I Double-Knockout Mice Fed Dietary Fat and Cholesterol

    PubMed Central

    Zabalawi, Manal; Bhat, Shaila; Loughlin, Tara; Thomas, Michael J.; Alexander, Eric; Cline, Mark; Bullock, Bill; Willingham, Mark; Sorci-Thomas, Mary G.

    2003-01-01

    Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr−/−) and apoA-I (apoA-I−/−) gene, LDLr−/−/apoA-I−/− or double-knockout mice. Gender- and age-matched LDLr−/−/apoA-I−/− mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr−/− mice or single-knockout mice. The LDLr−/− mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr−/−/apoA-I−/− mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr−/− and LDLr−/−/apoA-I−/− mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr−/− mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr−/− mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr−/−/apoA-I−/− mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr−/− mice showed similar aortic cholesterol levels to male LDLr−/−/apoA-I−/− mice despite a 4-fold higher VLDL/LDL concentration in the LDLr−/− mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr−/− and LDLr−/−/apoA-I−/− mice was compromised due to the loss of female LDLr−/−/apoA-I−/− mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr−/−/apoA-I−/− mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr−/− and LDLr−/−/apoA-I−/− mice showed

  3. Monitoring of Double Stud Wall Moisture Conditions in the Northeast

    SciTech Connect

    Ueno, K.

    2015-03-01

    Double-stud walls insulated with cellulose or low-density spray foam can have R-values of 40 or higher. However, double stud walls have a higher risk of interior-sourced condensation moisture damage, when compared with high-R approaches using exterior insulating sheathing.; Moisture conditions in double stud walls were monitored in Zone 5A (Massachusetts); three double stud assemblies were compared.

  4. Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study

    PubMed Central

    Coleman, James L. J.; Brennan, Karen; Ngo, Tony; Balaji, Poornima; Graham, Robert M.; Smith, Nicola J.

    2015-01-01

    As little as a decade ago, generation of a single knockout mouse line was an expensive and time-consuming undertaking available to relatively few researchers. The International Knockout Mouse Consortium, established in 2007, has revolutionized the use of such models by creating an open-access repository of embryonic stem (ES) cells that, through sequential breeding with first FLP1 recombinase and then Cre recombinase transgenic mice, facilitates germline global or conditional deletion of almost every gene in the mouse genome. In this Case Study, we describe our experience using the repository to create mouse lines for a variety of experimental purposes. Specifically, we discuss the process of obtaining germline transmission of two European Conditional Mouse Mutagenesis Program (EUCOMM) “knockout-first” gene targeted constructs and the advantages and pitfalls of using this system. We then outline our breeding strategy and the outcomes of our efforts to generate global and conditional knockouts and reporter mice for the genes of interest. Line maintenance, removal of recombinase transgenes, and cryopreservation are also considered. Our approach led to the generation of heterozygous knockout mice within 6 months of commencing breeding to the founder mice. By describing our experiences with the EUCOMM ES cells and subsequent breeding steps, we hope to assist other researchers with the application of this valuable approach to generating versatile knockout mouse lines. PMID:26175717

  5. Altered mnemonic functions and resistance to NMDA receptor antagonism by forebrain conditional knockout of glycine transporter 1

    PubMed Central

    Singer, Philipp; Yee, Benjamin K.; Feldon, Joram; Iwasato, Takuji; Itohara, Shigeyoshi; Grampp, Thomas; Prenosil, George; Benke, Dietmar; Möhler, Hanns; Boison, Detlev

    2009-01-01

    Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-D-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remains to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 down-regulation in the brain on behaviour and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated EPSC was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated. PMID:19332109

  6. Conditional Knockout in Mice Reveals the Critical Roles of Ppp2ca in Epidermis Development

    PubMed Central

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-01-01

    The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A) is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα) is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2caflox/flox transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2caflox/flox; Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2caflox/flox; Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2caflox/flox mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases. PMID:27213341

  7. Conditional Knockout in Mice Reveals the Critical Roles of Ppp2ca in Epidermis Development.

    PubMed

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-01-01

    The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A) is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα) is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2ca(flox/flox) transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2ca(flox/flox); Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2ca(flox/flox); Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2ca(flox/flox) mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases. PMID:27213341

  8. Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

    PubMed Central

    Migliarini, Sara; Pacini, Giulia; Pasqualetti, Massimo

    2015-01-01

    Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2null/null mice. Finally, we set up

  9. Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

    PubMed Central

    Liu, Ya-Wen; Surka, Mark C.; Schroeter, Thomas; Lukiyanchuk, Vasyl

    2008-01-01

    Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. PMID:18923138

  10. Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells.

    PubMed

    Liu, Ya-Wen; Surka, Mark C; Schroeter, Thomas; Lukiyanchuk, Vasyl; Schmid, Sandra L

    2008-12-01

    Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. PMID:18923138

  11. Virally-expressed connexin26 restores gap junction function in the cochlea of conditional Gjb2 knockout mice

    PubMed Central

    Yu, Qing; Wang, Yunfeng; Chang, Qing; Wang, Jianjun; Gong, Shushen; Li, Huawei; Lin, Xi

    2013-01-01

    Mutations in GJB2, which codes for the gap junction protein connexin26, are the most common causes of human nonsyndromic hereditary deafness. We inoculated modified adeno-associated viral vectors into the scala media of early postnatal conditional Gjb2 knockout mice to drive exogenous connexin26 expression. We found extensive virally-expressed connexin26 in cells lining the scala media, and intercellular gap junction network was re-established in the organ of Corti of mutant mouse cochlea. Widespread ectopic connexin26 expression neither formed ectopic gap junctions nor affected normal hearing thresholds in wild type mice, suggesting that autonomous cellular mechanisms regulate proper membrane trafficking of exogenously-expressed connexin26 and govern the functional manifestation of them. Functional recovery of gap-junction-mediated coupling among the supporting cells was observed. We found that both cell death in the organ of Corti and degeneration of spiral ganglion neurons in the cochlea of mutant mice were substantially reduced, although auditory brainstem responses did not show significant hearing improvement. This is the first report demonstrating that virally-mediated gene therapy restored extensive gap junction intercellular network among cochlear non-sensory cells in vivo. Such a treatment performed at early postnatal stages resulted in a partial rescue of disease phenotypes in the cochlea of the mutant mice. PMID:24225640

  12. Imbalanced lipid homeostasis in the conditional Dicer1 knockout mouse epididymis causes instability of the sperm membrane.

    PubMed

    Björkgren, Ida; Gylling, Helena; Turunen, Heikki; Huhtaniemi, Ilpo; Strauss, Leena; Poutanen, Matti; Sipilä, Petra

    2015-02-01

    During epididymal sperm maturation, the lipid content of the sperm membrane is modified, which facilitates sperm motility and fertility. However, little is known about the mechanisms regulating the maturation process. By generating a conditional knockout (cKO) of Dicer1 in the proximal part of the mouse epididymis, we studied the role of RNA interference in epididymal functions. The Dicer1 cKO epididymis displayed an altered lipid homeostasis associated with a 0.6-fold reduction in the expression of the gene elongation of very long chain fatty acids-like 2, an enzyme needed for production of long-chain polyunsaturated fatty acids (PUFAs). Furthermore, the expression of several factors involved in cholesterol synthesis was up-regulated. Accordingly, the Dicer1 cKO sperm membrane showed a 0.7-fold decrease in long-chain PUFAs, whereas the amount of cholesterol in acrosome-reacted sperm displayed a 1.7-fold increase. The increased cholesterol:PUFA ratio of the sperm membrane caused breakage of the neck and acrosome region and immotility of sperm. Dicer1 cKO mice sperm also displayed reduced ability to bind to and fertilize the oocyte in vitro. This study thus shows that Dicer1 is critical for lipid synthesis in the epididymis, which directly affects sperm membrane integrity and male fertility. PMID:25366345

  13. Conditional neuroligin-2 knockout in adult medial prefrontal cortex links chronic changes in synaptic inhibition to cognitive impairments.

    PubMed

    Liang, J; Xu, W; Hsu, Y-T; Yee, A X; Chen, L; Südhof, T C

    2015-07-01

    Abnormal activity in the medial prefrontal cortex (mPFC) is consistently observed in neuropsychiatric disorders, but the mechanisms involved remain unclear. Chronic aberrant excitation and/or inhibition of mPFC neurons were proposed to cause cognitive impairments. However, direct evidence for this hypothesis is lacking because it is technically challenging to control synaptic properties in a chronic and locally restricted, yet specific, manner. Here, we generated conditional knockout (cKO) mice of neuroligin-2 (Nlgn2), a postsynaptic cell-adhesion molecule of inhibitory synapses linked to neuropsychiatric disorders. cKO of Nlgn2 in adult mPFC rendered Nlgn2 protein undetectable after already 2-3 weeks, but induced major reductions in synaptic inhibition after only 6-7 weeks, and caused parallel impairments in anxiety, fear memory and social interaction behaviors. Moreover, cKO of Nlgn2 severely impaired behavioral stimulation of immediate-early gene expression in the mPFC, suggesting that chronic reduction in synaptic inhibition uncoupled the mPFC from experience-dependent inputs. Our results indicate that Nlgn2 is required for continuous maintenance of inhibitory synapses in the adult mPFC, and that chronic impairment of local inhibition disengages the mPFC from its cognitive functions by partially uncoupling the mPFC from experience-induced inputs. PMID:25824299

  14. An Altered Phenotype in a Conditional Knockout of Pitx2 in Extraocular Muscle

    PubMed Central

    Zhou, Yuefang; Cheng, Georgiana; Dieter, Lisa; Hjalt, Tord A.; Andrade, Francisco H.; Stahl, John S.; Kaminski, Henry J.

    2015-01-01

    Purpose To determine the temporal and spatial expression of Pitx2, a bicoid-like homeobox transcription factor, during postnatal development of mouse extraocular muscle and to evaluate its role in the growth and phenotypic maintenance of postnatal extraocular muscle. Methods Mouse extraocular muscles of different ages were examined for the expression of Pitx2 by RT-PCR, q-PCR, and immunostaining. A conditional mutant mouse strain, in which Pitx2 function is inactivated at postnatal day (P)0, was generated with a Cre-loxP strategy. Histology, immunostaining, realtime PCR, in vitro muscle contractility, and in vivo ocular motility were used to study the effect of Pitx2 depletion on extraocular muscle. Results All three Pitx2 isoforms were expressed by extraocular muscle and at higher levels than in other striated muscles. Immunostaining demonstrated the presence of Pitx2 mainly in extraocular muscle myonuclei. However, no obvious expression patterns were observed in terms of anatomic region (orbital versus global layer), innervation zone, or muscle fiber types. The mutant extraocular muscle had no obvious pathology but had altered muscle fiber sizes. Expression levels of myosin isoforms Myh1, Myh6, Myh7, and Myh13 were reduced, whereas Myh2, Myh3, Myh4, and Myh8 were not affected by postnatal loss of Pitx2. In vitro, Pitx2 loss made the extraocular muscles stronger, faster, and more fatigable. Eye movement recordings found saccades to have a lower peak velocity. Conclusions Pitx2 is important in maintaining the mature extraocular muscle phenotype and regulating the expression of critical contractile proteins. Modulation of Pitx2 expression can influence extraocular muscle function with long-term therapeutic implications. PMID:19407022

  15. Aged neuronal nitric oxide knockout mice show preserved olfactory learning in both social recognition and odor-conditioning tasks

    PubMed Central

    James, Bronwen M.; Li, Qin; Luo, Lizhu; Kendrick, Keith M.

    2015-01-01

    There is evidence for both neurotoxic and neuroprotective roles of nitric oxide (NO) in the brain and changes in the expression of the neuronal isoform of NO synthase (nNOS) gene occur during aging. The current studies have investigated potential support for either a neurotoxic or neuroprotective role of NO derived from nNOS in the context of aging by comparing olfactory learning and locomotor function in young compared to old nNOS knockout (nNOS−/−) and wildtype control mice. Tasks involving social recognition and olfactory conditioning paradigms showed that old nNOS−/− animals had improved retention of learning compared to similar aged wildtype controls. Young nNOS−/− animals showed superior reversal learning to wildtypes in a conditioned learning task, although their performance was weakened with age. Interestingly, whereas young nNOS−/− animals were impaired in long term memory for social odors compared to wildtype controls, in old animals this pattern was reversed, possibly indicating beneficial compensatory changes influencing olfactory memory may occur during aging in nNOS−/− animals. Possibly such compensatory changes may have involved increased NO from other NOS isoforms since the memory deficit in young nNOS−/− animals could be rescued by the NO-donor, molsidomine. Both nNOS−/− and wildtype animals showed an age-associated decline in locomotor activity although young nNOS−/− animals were significantly more active than wildtypes, possibly due to an increased interest in novelty. Overall our findings suggest that lack of NO release via nNOS may protect animals to some extent against age-associated cognitive decline in memory tasks typically involving olfactory and hippocampal regions, but not against declines in reversal learning or locomotor activity. PMID:25870540

  16. Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis.

    PubMed

    Valenzuela, Nicolas; Fan, Qiying; Fa'ak, Faisal; Soibam, Benjamin; Nagandla, Harika; Liu, Yu; Schwartz, Robert J; McConnell, Bradley K; Stewart, M David

    2016-03-01

    HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. PMID:26935106

  17. Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis

    PubMed Central

    Valenzuela, Nicolas; Fan, Qiying; Fa'ak, Faisal; Soibam, Benjamin; Nagandla, Harika; Liu, Yu; Schwartz, Robert J.; McConnell, Bradley K.; Stewart, M. David

    2016-01-01

    ABSTRACT HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. PMID:26935106

  18. Application of the double absorbing boundary condition in seismic modeling

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Xiang-Yang; Chen, Shuang-Quan

    2015-03-01

    We apply the newly proposed double absorbing boundary condition (DABC) (Hagstrom et al., 2014) to solve the boundary reflection problem in seismic finite-difference (FD) modeling. In the DABC scheme, the local high-order absorbing boundary condition is used on two parallel artificial boundaries, and thus double absorption is achieved. Using the general 2D acoustic wave propagation equations as an example, we use the DABC in seismic FD modeling, and discuss the derivation and implementation steps in detail. Compared with the perfectly matched layer (PML), the complexity decreases, and the stability and flexibility improve. A homogeneous model and the SEG salt model are selected for numerical experiments. The results show that absorption using the DABC is considerably improved relative to the Clayton-Engquist boundary condition and nearly the same as that in the PML.

  19. A facile one-step strategy for the generation of conditional knockout mice to explore the role of Notch1 in oroesophageal tumorigenesis.

    PubMed

    Mandasari, Masita; Sawangarun, Wanlada; Katsube, Ken-ichi; Kayamori, Kou; Yamaguchi, Akira; Sakamoto, Kei

    2016-01-15

    NOTCH1 plays an important role in epithelial differentiation and carcinogenesis. To investigate the impact of Notch1 inactivation in oroesophageal epithelium, we generated conditional knockout (cKO) mice, using a combined construct which induces the expression of single guide RNA targeting Notch1 and Cas9 by the KRT14 promoter. The cKO mice exhibited patchy hair loss and multiple NOTCH1-negative areas in the tongue epithelium, indicative of heterogeneous knockout. The cKO mice showed susceptibility to esophageal tumorigenesis, underscoring Notch1 as a tumor suppressor. Our one-step strategy for generation of cKO mice provides a versatile method to examine a gene function in vivo. PMID:26682927

  20. Distribution of selected elements in atherosclerotic plaques of apoE/LDLR-double knockout mice subjected to dietary and pharmacological treatments

    NASA Astrophysics Data System (ADS)

    Gajda, Mariusz; Kowalska, Joanna; Banaś, Agnieszka; Banaś, Krzysztof; Kwiatek, Wojciech M.; Kostogrys, Renata B.; Mateuszuk, łukasz; ChŁopicki, Stefan; Litwin, Jan A.; Appel, Karen

    2011-10-01

    Gene-targeted, apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice represent a new animal model that displays severe hyperlipidemia and atherosclerosis. The aim of the present study was to show changes in histomorphology and in distribution of selected elements in atherosclerotic plaques of apoE/LDLR -/- mice fed egg-rich proatherosclerotic diet (5% egg-yolk lyophilisate) supplemented or not with perindopril (inhibitor of angiotensin converting enzyme; 2 mg/kg b.w.). Synchrotron radiation micro-X-ray fluorescence spectrometry was combined with histological stainings to determine distribution and concentration of trace and essential elements in atherosclerotic lesions. More advanced atherosclerotic lesions expressed by total area occupied by lipids (oil red-O staining) and by macrophages (CD68 immunohistochemistry) were observed in animals fed egg-rich diet. The perindopril treatment attenuated these effects. No significant differences were observed in the number of intimal smooth muscle cells (smooth muscle actin immunohistochemistry). In animals fed egg-rich diet significantly higher concentrations of Ca and significantly lower contents of S, Cl, , Fe, Cu, Zn and Se in atheromas were seen in comparison to chow diet-fed animals. After pharmacological treatment, concentrations of S, Cl, Fe, Cu, Zn and Se showed the tendency to achieve levels like in animals fed normal diet. K level differed only in group treated with perindopril. Concentration of P did not significantly vary in all experimental groups. Perindopril showed its potency to reduce atherosclerosis, as estimated by the size of the atheroma and content of pro- and antiatherogenic elements.

  1. AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

    PubMed Central

    Boye, Sanford L.; Peshenko, Igor V.; Huang, Wei Chieh; Min, Seok Hong; McDoom, Issam; Kay, Christine N.; Liu, Xuan; Dyka, Frank M.; Foster, Thomas C.; Umino, Yumiko; Karan, Sukanya; Jacobson, Samuel G.; Baehr, Wolfgang; Dizhoor, Alexander; Hauswirth, William W.

    2013-01-01

    Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1. PMID:23210611

  2. Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice.

    PubMed

    Yadav, Manisha C; Bottini, Massimo; Cory, Esther; Bhattacharya, Kunal; Kuss, Pia; Narisawa, Sonoko; Sah, Robert L; Beck, Laurent; Fadeel, Bengt; Farquharson, Colin; Millán, José Luis

    2016-06-01

    We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that ∼80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to ∼50% for the Phospho1(-/-) MVs and ∼25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and

  3. Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

    PubMed

    Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

    2016-07-01

    In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. PMID:26980143

  4. Similar phenotypes of Girdin germ-line and conditional knockout mice indicate a crucial role for Girdin in the nestin lineage.

    PubMed

    Asai, Masato; Asai, Naoya; Murata, Ayana; Yokota, Hirofumi; Ohmori, Kenji; Mii, Shinji; Enomoto, Atsushi; Murakumo, Yoshiki; Takahashi, Masahide

    2012-10-01

    Girdin is an Akt substrate and actin-binding protein. Mice with germ-line deletions of Girdin (a non-conditional knockout, (ncKO)) exhibit complete postnatal lethality accompanied by growth retardation and neuronal cell migration defects, which results in hypoplasia of the olfactory bulb and granule cell dispersion in the dentate gyrus. However, the physiological and molecular abnormalities in Girdin ncKO mice are not fully understood. In this study, we first defined the distribution of Girdin in neonates (P1) and adults (6months or older) using β-galactosidase activity in tissues from ncKO mice. The results indicate that Girdin is expressed throughout the nervous system (brain, spinal cord, enteric and autonomic nervous systems). In addition, β-galactosidase activity was detected in non-neural tissues, particularly in tissues with high tensile force, such as tendons, heart valves, and skeletal muscle. In order to identify the cellular population where the Girdin ncKO phenotype originates, newly generated Girdin flox mice were crossed with nestin promoter-driven Cre transgenic mice to obtain Girdin conditional knockout (cKO) mice. The phenotype of Girdin cKO mice was almost identical to ncKO mice, including postnatal lethality, growth retardation and decreased neuronal migration. Our findings indicate that loss of Girdin in the nestin cell lineage underlies the phenotype of Girdin ncKO mice. PMID:22974978

  5. Junction conditions in quadratic gravity: thin shells and double layers

    NASA Astrophysics Data System (ADS)

    Reina, Borja; Senovilla, José M. M.; Vera, Raül

    2016-05-01

    The junction conditions for the most general gravitational theory with a Lagrangian containing terms quadratic in the curvature are derived. We include the cases with a possible concentration of matter on the joining hypersurface—termed as thin shells, domain walls or braneworlds in the literature—as well as the proper matching conditions where only finite jumps of the energy-momentum tensor are allowed. In the latter case we prove that the matching conditions are more demanding than in general relativity. In the former case, we show that generically the shells/domain walls are of a new kind because they possess, in addition to the standard energy-momentum tensor, a double layer energy-momentum contribution which actually induces an external energy flux vector and an external scalar pressure/tension on the shell. We prove that all these contributions are necessary to make the entire energy-momentum tensor divergence-free, and we present the field equations satisfied by these energy-momentum quantities. The consequences of all these results are briefly analyzed.

  6. Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts.

    PubMed

    Morán-Salvador, Eva; López-Parra, Marta; García-Alonso, Verónica; Titos, Esther; Martínez-Clemente, Marcos; González-Périz, Ana; López-Vicario, Cristina; Barak, Yaacov; Arroyo, Vicente; Clària, Joan

    2011-08-01

    Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and β-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease. PMID:21507897

  7. Altered mnemonic functions and resistance to N-METHYL-d-Aspartate receptor antagonism by forebrain conditional knockout of glycine transporter 1.

    PubMed

    Singer, P; Yee, B K; Feldon, J; Iwasato, T; Itohara, S; Grampp, T; Prenosil, G; Benke, D; Möhler, H; Boison, D

    2009-06-30

    Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated. PMID:19332109

  8. Knockout of p11 attenuates the acquisition and reinstatement of cocaine conditioned place preference in male but not in female mice.

    PubMed

    Thanos, Panayotis K; Malave, Lauren; Delis, Foteini; Mangine, Paul; Kane, Katie; Grunseich, Adam; Vitale, Melissa; Greengard, Paul; Volkow, Nora D

    2016-07-01

    Cocaine's enhancement of dopamine signaling is crucial for its rewarding effects but its serotonergic effects are also relevant. Here we examined the role of the protein p11, which recruits serotonin 5HT1B and 5HT4 receptors to the cell surface, in cocaine reward. For this purpose we tested wild-type (WT) and p11 knockout (KO) male and female mice for cocaine conditioned place preference (CPP) and its cocaine-induced reinstatement at different abstinence times, after 8 days of extinction and 28 days of being home-caged. All mice showed significant cocaine CPP. Among males, p11KO showed lower CPP than WT; this difference was also evident after 28 days of home-cage abstinence. In contrast, in females there were no CPP differences between p11KO and WT mice at any time point tested. Cocaine priming after the 28-day home-cage abstinence period also resulted in lower cocaine conditioned motor activity in both male and female p11KO mice. These results suggest that cocaine CPP and its persistence during extinction and reinstatement are modulated in a sex-differentiated manner by p11. The lack of protein p11 confers protection from CPP on male, but not female mice, immediately after cocaine conditioning as well as after prolonged abstinence, but not after short-term withdrawal. Synapse 70:293-301, 2016. © 2016 Wiley Periodicals, Inc. PMID:26990537

  9. Monitoring of Double-Stud Wall Moisture Conditions in the Northeast

    SciTech Connect

    Ueno, K.

    2015-03-01

    Double-stud walls insulated with cellulose or low-density spray foam can have R-values of 40 or higher. However, double-stud walls have a higher risk of interior-sourced condensation moisture damage when compared with high-R approaches using exterior insulating sheathing. Moisture conditions in double-stud walls were monitored in Zone 5A (Massachusetts); three double-stud assemblies were compared.

  10. Phosphodiesterase 1B differentially modulates the effects of methamphetamine on locomotor activity and spatial learning through DARPP32-dependent pathways: evidence from PDE1B-DARPP32 double-knockout mice.

    PubMed

    Ehrman, L A; Williams, M T; Schaefer, T L; Gudelsky, G A; Reed, T M; Fienberg, A A; Greengard, P; Vorhees, C V

    2006-10-01

    Mice lacking phosphodiesterase 1B (PDE1B) exhibit an exaggerated locomotor response to D-methamphetamine and increased in vitro phosphorylation of DARPP32 (dopamine- and cAMP-regulated phosphoprotein, M r 32 kDa) at Thr34 in striatal brain slices treated with the D1 receptor agonist, SKF81297. These results indicated a possible regulatory role for PDE1B in pathways involving DARPP32. Here, we generated PDE1B x DARPP32 double-knockout (double-KO) mice to test the role of PDE1B in DARPP32-dependent pathways in vivo. Analysis of the response to d-methamphetamine on locomotor activity showed that the hyperactivity experienced by PDE1B mutant mice was blocked in PDE1B-/- x DARPP32-/- double-KO mice, consistent with participation of PDE1B and DARPP32 in the same pathway. Further behavioral testing in the elevated zero-maze revealed that DARPP32-/- mice showed a less anxious phenotype that was nullified in double-mutant mice. In contrast, in the Morris water maze, double-KO mice showed deficits in spatial reversal learning not observed in either single mutant compared with wild-type mice. The data suggest a role for PDE1B in locomotor responses to psychostimulants through modulation of DARPP32-dependent pathways; however, this modulation does not necessarily impact other behaviors, such as anxiety or learning. Instead, the phenotype of double-KOs observed in these latter tasks may be mediated through independent pathways. PMID:17010100

  11. Conditional Knockout of Tumor Overexpressed Gene in Mouse Neurons Affects RNA Granule Assembly, Granule Translation, LTP and Short Term Habituation

    PubMed Central

    Barbarese, Elisa; Ifrim, Marius F.; Hsieh, Lawrence; Guo, Caiying; Tatavarty, Vedakumar; Maggipinto, Michael J.; Korza, George; Tutolo, Jessica W.; Giampetruzzi, Anthony; Le, Hien; Ma, Xin-Ming; Levine, Eric; Bishop, Brian; Kim, Duck O.; Kuwada, Shigeyuki; Carson, John H.

    2013-01-01

    In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG) is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP) A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs) present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO) in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC) mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP) are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior. PMID:23936366

  12. Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii.

    PubMed

    Rommereim, Leah M; Bellini, Valeria; Fox, Barbara A; Pètre, Graciane; Rak, Camille; Touquet, Bastien; Aldebert, Delphine; Dubremetz, Jean-François; Cesbron-Delauw, Marie-France; Mercier, Corinne; Bzik, David J

    2016-01-01

    Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection. PMID:27458822

  13. Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

    PubMed Central

    Fox, Barbara A.; Pètre, Graciane; Rak, Camille; Touquet, Bastien; Aldebert, Delphine; Dubremetz, Jean-François; Cesbron-Delauw, Marie-France; Mercier, Corinne; Bzik, David J.

    2016-01-01

    Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection. PMID:27458822

  14. Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

    PubMed Central

    Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

    2013-01-01

    Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

  15. Conditional knockout of the leptin receptor in the colonic epithelium revealed the local effects of leptin receptor signaling in the progression of colonic tumors in mice.

    PubMed

    Higurashi, Takuma; Endo, Hiroki; Uchiyama, Takashi; Uchiyama, Shiori; Yamada, Eiji; Ohkubo, Hidenori; Sakai, Eiji; Takahashi, Hirokazu; Maeda, Shin; Wada, Koichiro; Natsumeda, Yutaka; Hippo, Yoshitaka; Nakajima, Atsushi; Nakagama, Hitoshi

    2014-09-01

    Leptin, secreted by the adipose tissue and known to be related to obesity, is considered to be involved in the onset and progression of colorectal cancer. However, the exact role of leptin in colorectal carcinogenesis is still unclear, as several controversial reports have been published on the various systemic effects of leptin. The aim of this study was to clarify the local and precise roles of leptin receptor (LEPR)-mediated signaling in colonic carcinogenesis using intestinal epithelium-specific LEPRb conditional knockout (cKO) mice. We produced and used colonic epithelium-specific LEPRb cKO mice to investigate the carcinogen-induced formation of aberrant crypt foci (ACF) and tumors in the colon, using their littermates as control. There were no differences in the body weight or systemic condition between the control and cKO mice. The tumor sizes and number of large-sized tumors were significantly lower in the cKO mice as compared with those in the control mice. On the other hand, there was no significant difference in the proliferative activity of the normal colonic epithelial cells or ACF formation between the control and cKO mice. In the control mice, marked increase of the LEPRb expression level was observed in the colonic tumors as compared with that in the normal epithelium; furthermore, signal transducer and activator of transcription (STAT3) was activated in the tumor cells. These findings suggest that STAT3 is one of the important molecules downstream of LEPRb, and LEPRb/STAT3 signaling controls tumor cell proliferation. We demonstrated the importance of local/regional LEPR-mediated signaling in colorectal carcinogenesis. PMID:24958593

  16. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

    PubMed

    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. PMID:26595646

  17. Host Susceptibility to Brucella abortus Infection Is More Pronounced in IFN-γ knockout than IL-12/β2-Microglobulin Double-Deficient Mice

    PubMed Central

    Brandão, Ana Paula M. S.; Oliveira, Fernanda S.; Carvalho, Natalia B.; Vieira, Leda Q.; Azevedo, Vasco; Macedo, Gilson C.; Oliveira, Sergio C.

    2012-01-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. IFN-γ, IL-12, and CD8+ T lymphocytes are important components of host immune responses against B. abortus. Herein, IFN-γ and IL-12/β2-microglobulin (β2-m) knockout mice were used to determine whether CD8+ T cells and IL-12-dependent IFN-γ deficiency would be more critical to control B. abortus infection compared to the lack of endogenous IFN-γ. At 1 week after infection, IFN-γ KO and IL-12/β2-m KO mice showed increased numbers of bacterial load in spleens; however, at 3 weeks postinfection (p.i.), only IFN-γ KO succumbed to Brucella. All IFN-γ KO had died at 16 days p.i. whereas death within the IL-12/β2-m KO group was delayed and occurred at 32 days until 47 days postinfection. Susceptibility of IL-12/β2-m KO animals to Brucella was associated to undetectable levels of IFN-γ in mouse splenocytes and inability of these cells to lyse Brucella-infected macrophages. However, the lack of endogenous IFN-γ was found to be more important to control brucellosis than CD8+ T cells and IL-12-dependent IFN-γ deficiencies. PMID:22194770

  18. How to impose initial conditions for QCD evolution of double parton distributions?

    NASA Astrophysics Data System (ADS)

    Golec-Biernat, Krzysztof; Lewandowska, Emilia

    2014-07-01

    Double parton distribution functions are used in the QCD description of double parton scattering. The double parton distributions evolve with hard scales through QCD evolution equations which obey nontrivial momentum and valence quark number sum rules. We describe an attempt to construct initial conditions for the evolution equations which exactly fulfill these sum rules and discuss its shortcomings. We also discuss the factorization of the double parton distributions into a product of two single parton distribution functions at small values of the parton momentum fractions.

  19. Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

    PubMed

    Amjad, Asma; Hernandez-Clavijo, Andres; Pifferi, Simone; Maurya, Devendra Kumar; Boccaccio, Anna; Franzot, Jessica; Rock, Jason; Menini, Anna

    2015-04-01

    Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca(2+) concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca(2+) in isolated mouse vomeronasal sensory neurons in the absence of intracellular K(+). In whole-cell recordings, the average current in 1.5 µM Ca(2+) and symmetrical Cl(-) was -382 pA at -100 mV. Ion substitution experiments and partial blockade by commonly used Cl(-) channel blockers indicated that Ca(2+) activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca(2+)-activated Cl(-) channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca(2+)-activated Cl(-) channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca(2+)-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca(2+)-activated Cl(-) currents in mouse vomeronasal sensory neurons. PMID:25779870

  20. Double-Shell Tank Construction: Extent of Condition

    SciTech Connect

    Venetz, Theodore J.; Gunter, Jason R.

    2014-05-13

    This presentation covers: quick recap of Hanford DSTs and the contribution of construction difficulties which led to the leak in tank AY-102; approach to Extent of Condition reviews; typical DST construction sequence; presentation of construction information resulting from extent of condition reviews of other DST farms with comparison to tank AY-102; and overall conclusion and impact of issues on the other DST tank farms.

  1. C-Terminal-Truncated Microdystrophin Recruits Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double-Knockout Mice

    PubMed Central

    Yue, Yongping; Liu, Mingju; Duan, Dongsheng

    2007-01-01

    C-terminal-truncated (ΔC) microdystrophin is being developed for Duchenne muscular dystrophy gene therapy. Encouraging results have been achieved in the mdx mouse model. Unfortunately, mdx mice do not display the same phenotype as human patients. Evaluating ΔC microdystrophin in a symptomatic model will be of significant relevance to human trials. Utrophin/dystrophin double-knockout (u-dko) mice were developed to model severe dystrophic changes in human patients. In this study we evaluated the therapeutic effect of the ΔR4-R23/ΔC microdystrophin gene (ΔR4/ΔC) after serotype-6 adeno-associated virus-mediated gene transfer in neonatal u-dko muscle. At 2 months after gene transfer, the percentage of centrally nucleated myofiber was reduced from 89.2 to 3.4% and muscle weight was normalized. Furthermore, we have demonstrated for the first time that ΔC microdystrophin can eliminate interstitial fibrosis and macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. Interestingly neuronal nitric oxide synthase was not restored. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch- and tetanic-specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the ΔR4/ΔC microgene holds great promise in preventing muscular dystrophy. PMID:16563874

  2. 13C nuclear magnetic resonance analysis of glucose and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum.

    PubMed Central

    Ferain, T; Schanck, A N; Delcour, J

    1996-01-01

    We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol. PMID:8955418

  3. Double P2X2/P2X3 Purinergic Receptor Knockout Mice Do Not Taste NaCl or the Artificial Sweetener SC45647

    PubMed Central

    Eddy, Meghan C.; Eschle, Benjamin K.; Barrows, Jennell; Hallock, Robert M.; Finger, Thomas E.

    2009-01-01

    The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3Dbl−/−) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495–1499). Here, we compare the ability of P2X2/P2X3Dbl−/− mice and P2X2/P2X3Dbl+/+ wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3Dbl−/− mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3Dbl−/− mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3Dbl−/− mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3Dbl−/− mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3Dbl−/− mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances. PMID:19833661

  4. Mechanisms in Knockout Reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-06-01

    We report the first detailed study of the relative importance of the stripping and diffraction mechanisms involved in nucleon knockout reactions, by the use of a coincidence measurement of the residue and fast proton following one-proton knockout reactions. The measurements used the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results for the reactions Be9(C9,B8+X)Y and Be9(B8,Be7+X)Y are presented and compared with theoretical predictions for the two reaction mechanisms calculated using the eikonal model. The data show a clear distinction between the stripping and diffraction mechanisms and the measured relative proportions are very well reproduced by the reaction theory. This agreement adds support to the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes.

  5. Boundary conditions and the generalized metric formulation of the double sigma model

    NASA Astrophysics Data System (ADS)

    Ma, Chen-Te

    2015-09-01

    Double sigma model with strong constraints is equivalent to the ordinary sigma model by imposing a self-duality relation. The gauge symmetries are the diffeomorphism and one-form gauge transformation with the strong constraints. We consider boundary conditions in the double sigma model from three ways. The first way is to modify the Dirichlet and Neumann boundary conditions with a fully O (D, D) description from double gauge fields. We perform the one-loop β function for the constant background fields to find low-energy effective theory without using the strong constraints. The low-energy theory can also have O (D, D) invariance as the double sigma model. The second way is to construct different boundary conditions from the projectors. The third way is to combine the antisymmetric background field with field strength to redefine an O (D, D) generalized metric. We use this generalized metric to reconstruct a consistent double sigma model with the classical and quantum equivalence.

  6. Inhibition of intestinal absorption of cholesterol by ezetimibe or bile acids by SC-435 alters lipoprotein metabolism and extends the lifespan of SR-BI/apoE double knockout mice.

    PubMed

    Braun, Anne; Yesilaltay, Ayce; Acton, Susan; Broschat, Kay O; Krul, Elaine S; Napawan, Nida; Stagliano, Nancy; Krieger, Monty

    2008-05-01

    SR-BI/apoE double knockout (dKO) mice exhibit many features of human coronary heart disease (CHD), including hypercholesterolemia, occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, cardiac dysfunction and premature death. Ezetimibe is a FDA-approved, intestinal cholesterol absorption inhibitor that lowers plasma LDL cholesterol in humans and animals and inhibits aortic root atherosclerosis in apoE KO mice, but has not been proven to reduce CHD. Three-week-ezetimibe treatment of dKO mice (0.005% (w/w) in standard chow administered from weaning) resulted in a 35% decrease in cholesterol in IDL/LDL-size lipoproteins, but not in VLDL- and HDL-size lipoproteins. Ezetimibe treatment significantly reduced aortic root (57%) and coronary arterial (68%) atherosclerosis, cardiomegaly (24%) and cardiac fibrosis (57%), and prolonged the lives of the mice (27%). This represents the first demonstration of beneficial effects of ezetimibe treatment on CHD. The dKO mice were similarly treated with SC-435 (0.01% (w/w)), an apical sodium codependent bile acid transporter (ASBT) inhibitor, that blocks intestinal absorption of bile acids, lowers plasma cholesterol in animals, and reduces aortic root atherosclerosis in apoE KO mice. The effects of SC-435 treatment were similar to those of ezetimibe: 37% decrease in ILD/LDL-size lipoprotein cholesterol and 57% prolongation in median lifespan. Thus, inhibition of intestinal absorption of either cholesterol (ezetimibe) or bile acids (SC-435) significantly reduced plasma IDL/LDL-size lipoprotein cholesterol levels and improved survival of SR-BI/apoE dKO mice. The SR-BI/apoE dKO murine model of atherosclerotic occlusive, arterial CHD appears to provide a useful system to evaluate compounds that modulate cholesterol homeostasis and atherosclerosis. PMID:18054357

  7. Technology Solutions Case Study: Monitoring of Double Stud Wall Moisture Conditions in the Northeast, Devens, Massachusetts

    SciTech Connect

    2015-03-01

    Double stud walls have a higher risk of interior-sourced condensation moisture damage when compared with high-R approaches using exterior insulating sheathing. In this project, Building Science Corporation monitored moisture conditions in double-stud walls from 2011 through 2014 at a new production house located in Devens, Massachusetts. The builder, Transformations, Inc., has been using double-stud walls insulated with 12 in. of open cell polyurethane spray foam (ocSPF); however, the company has been considering a change to netted and blown cellulose insulation for cost reasons. Cellulose is a common choice for double-stud walls because of its lower cost (in most markets). However, cellulose is an air-permeable insulation, unlike spray foams, which increases interior moisture risks. The team compared three double-stud assemblies: 12 in. of ocSPF, 12 in. of cellulose, and 5-½ in. of ocSPF at the exterior of a double-stud wall (to approximate conventional 2 × 6 wall construction and insulation levels, acting as a control wall). These assemblies were repeated on the north and south orientations, for a total of six assemblies.

  8. Conditions for establishing quasistable double layers in the Earth's auroral upward current region

    SciTech Connect

    Main, D. S.; Newman, D. L.; Ergun, R. E.

    2010-12-15

    The strength and stability of simulated double layers at the ionosphere-auroral cavity boundary have been studied as a function of cold ionospheric electron temperature and density. The simulations are performed with an open boundary one-dimensional particle-in- cell (PIC) simulation and are initialized by imposing a density cavity within the simulation domain. The PIC simulation includes H{sup +} and O{sup +} ion beams, a hot H{sup +} background population, cold ionospheric electrons, and a hot electron population. It is shown that a double layer remains quasistable for a variety of initial conditions and plasma parameters. The average potential drop of the double layer is found to increase as the cold electron temperature decreases. However, in terms of cold electron density, the average potential drop of the double layer is found to increase up to some critical cold electron density and decreases above this value. Comparisons with FAST observations are made and agreement is found between simulation results and observations in the shape and width of the double layer. This study helps put a constraint on the plasma conditions in which a DL can be expected to form and remain quasistable.

  9. Double Dissociation of Conditioning and Declarative Knowledge Relative to the Amygdala and Hippocampus in Humans

    NASA Astrophysics Data System (ADS)

    Bechara, Antoine; Tranel, Daniel; Damasio, Hanna; Adolphs, Ralph; Rockland, Charles; Damasio, Antonio R.

    1995-08-01

    A patient with selective bilateral damage to the amygdala did not acquire conditioned autonomic responses to visual or auditory stimuli but did acquire the declarative facts about which visual or auditory stimuli were paired with the unconditioned stimulus. By contrast, a patient with selective bilateral damage to the hippocampus failed to acquire the facts but did acquire the conditioning. Finally, a patient with bilateral damage to both amygdala and hippocampal formation acquired neither the conditioning nor the facts. These findings demonstrate a double dissociation of conditioning and declarative knowledge relative to the human amygdala and hippocampus.

  10. Building America Case Study: Monitoring of Double Stud Wall Moisture Conditions in the Northeast, Devens, Massachusetts (Fact Sheet)

    SciTech Connect

    Not Available

    2015-03-01

    Double-stud walls insulated with cellulose or low-density spray foam can have R-values of 40 or higher. However, double stud walls have a higher risk of interior-sourced condensation moisture damage, when compared with high-R approaches using exterior insulating sheathing. Moisture conditions in double stud walls were monitored in Zone 5A (Massachusetts); three double stud assemblies were compared.

  11. Mechanisms in knockout reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-10-01

    We report on the first detailed study of the mechanisms involved in knockout reactions, via a coincidence measurement of the residue and fast proton in one-proton knockout reactions, using the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results on the reactions ^9Be(^9C,^8B+X)Y and ^9Be(^8B,^7Be+X)Y are presented. They are compared with theoretical predictions for both the diffraction (elastic breakup) and stripping (inelastic breakup) reaction mechanisms, as calculated in the eikonal model. The data shows a clear distinction between the two reaction mechanisms, and the observed respective proportions are very well reproduced by the reaction theory. This agreement supports the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes. In particular, this add considerable support to the use of the eikonal model as a quantitative tool, able, for example, to determine single-particle spectroscopic strengths in rare isotopes.

  12. Effects of D-4F on Vasodilation and Vessel Wall Thickness in Hypercholesterolemic LDL Receptor Null and LDL receptor/ApoA-I Double Knockout Mice on Western Diet

    PubMed Central

    Ou, Jingsong; Wang, Jingli; Xu, Hao; Ou, Zhijun; Sorci-Thomas, Mary G.; Jones, Deron W.; Signorino, Paul; Densmore, John C.; Kaul, Sushma; Oldham, Keith T.; Pritchard, Kirkwood A.

    2005-01-01

    Previously we showed L-4F, a novel apolipoprotein A-I (apoA-I) mimetic, improved vasodilation in two dissimilar models of vascular disease; hypercholesterolemic low-density lipoprotein (LDL) receptor null (Ldlr −/−) mice and transgenic sickle cell disease mice. Here we determine the mechanisms by which D-4F improves vasodilation and arterial wall thickness in hypercholesterolemic Ldlr −/− mice and Ldlr −/−/apoA-I null (apoA-I −/−), double knockout mice. Ldlr −/− and Ldlr −/−/apoA-I −/− mice were fed western diet (WD) ± D-4F. Oral D-4F restored endothelium- and eNOS-dependent vasodilation in direct relationship to duration of treatments and reduced wall thickness in as little as 2 weeks in vessels with pre-existing disease in Ldlr −/− mice. D-4F had no effect on total or HDL cholesterol concentrations but reduced proinflammatory HDL levels. D-4F had no effect on plasma myeloperoxidase (MPO) concentrations but reduced MPO association with apoA-I as well as 3-nitrotyrosine in apoA-I. D-4F increased endothelium- and eNOS-dependent vasodilation in Ldlr −/−/apoA-I −/− mice but did not reduce wall thickness as it had in Ldlr −/− mice. Vascular endothelial cells were treated with 22-hydroxycholesterol (22-OHC) ± L-4F. 22-OHC decreased nitric oxide (•NO) and increased superoxide anion (O2 •−) production and increased ABCA-1 and collagen expression. L-4F restored •NO and O2 •− balance, had little effect on ABCA-1 expression but reduced collagen expression. These data demonstrate that although D-4F restores vascular endothelial cell and eNOS function to increase vasodilation, HDL containing apoA-I, or at least some critical concentration of the anti-atherogenic lipoprotein, is required for D-4F to decrease vessel wall thickness. PMID:16224061

  13. The contribution of single and double cones to spectral sensitivity in budgerigars during changing light conditions.

    PubMed

    Lind, Olle; Chavez, Johanna; Kelber, Almut

    2014-03-01

    Bird colour vision is mediated by single cones, while double cones and rods mediate luminance vision in bright and dim light, respectively. In daylight conditions, birds use colour vision to discriminate large objects such as fruit and plumage patches, and luminance vision to detect fine spatial detail and motion. However, decreasing light intensity favours achromatic mechanisms and eventually, in dim light, luminance vision outperforms colour vision in all visual tasks. We have used behavioural tests in budgerigars (Melopsittacus undulatus) to investigate how single cones, double cones and rods contribute to spectral sensitivity for large (3.4°) static monochromatic stimuli at light intensities ranging from 0.08 to 63.5 cd/m². We found no influences of rods at any intensity level. Single cones dominate the spectral sensitivity function at intensities above 1.1 cd/m², as predicted by a receptor noise-limited colour discrimination model. Below 1.1 cd/m², spectral sensitivity is lower than expected at all wavelengths except 575 nm, which corresponds to double cone function. We suggest that luminance vision mediated by double cones restores visual sensitivity when single cone sensitivity quickly decreases at light intensities close to the absolute threshold of colour vision. PMID:24366429

  14. Deformation of double emulsions under conditions of flow cytometry hydrodynamic focusing.

    PubMed

    Ma, Shaohua; Huck, Wilhelm T S; Balabani, Stavroula

    2015-11-21

    Water-in-oil-in-water (w/o/w) microfluidics double emulsions offer a new route to compartmentalise reagents into isolated aqueous microenvironments while maintaining an aqueous carrier fluid phase; this enables compatibility with commercial flow cytometry systems such as fluorescence-activated cell sorting (FACS). Double emulsion (inner core) deformation under hydrodynamic focusing conditions that mimic the environment double emulsions experience in flow cytometry applications is of particular importance for droplet stability and cell viability. This paper reports on an experimental study of the dynamic deformation of aqueous cores of w/o/w double emulsions under hydrodynamic focusing, with the sheath flow directed at 45° to the sample flow. A number of factors affecting the inner core deformation and recovery were examined. Deformation was found to depend significantly on the core or shell viscosity, the droplet-to-sheath flow velocity ratio, and core and shell sizes. Core deformation was found to depend more on the type of surfactant rather concentration with high molecular weight surfactant exhibiting a negligible effect on deformation whereas low molecular weight surfactant enhancing deformation at low concentrations due to their lateral mobility at the interface. PMID:26394745

  15. TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR-MALDESI imaging of lipids and LC-MS/MS label-free proteomics.

    PubMed

    Loziuk, Philip; Meier, Florian; Johnson, Caroline; Ghashghaei, H Troy; Muddiman, David C

    2016-05-01

    Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of "omics" and "big data." Here, we provide an integrated approach for systematic analysis of the "lipidome" in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos. Graphical abstract TransOmic Analysis of Dorsolateral cortices of Sp2 conditional Knockout mouse embryos. Target tissue was extracted by laser microdissection and analyzed by LC-MS/MS label- free quantitative proteomics. In parallel, lipid imaging of these tissues was performed by conventional IR-MALDESI and Agdoped IR-MALDESI imaging. These data were then integrated to obtain insight into lipid pathways altered by presence or absence of transcription factor Specificity Protein 2. PMID:26942738

  16. Risk assessment under current and double CO{sub 2} conditions for United States wheat yields

    SciTech Connect

    Barry, T.A.; Geng, S.

    1995-03-01

    There are at least two problems in quantifying weather related risks associated with crop yields. First, long-term data are generally not available. Second, even if the data were available, they were frequently confounded with factors other than weather conditions. In this study the authors have used a simulation approach to quantify two types of risks associated with cropping systems under current and double CO{sub 2} scenarios. The first order risk is defined as the probability of crop failure in a particular environment. The second order risk is the degree of uncertainty of maintaining the mean yield for a growing condition (variance of yield). Results show that, overall, with the present cropping systems the first order risk increased by 20% with doubling of CO{sub 2}. Second order risk increased at locations that presently have the highest mean wheat yields in the Great Plains. Second order risk decreased at locations that currently produce the majority of wheat in the Great Plains. However, at those locations the mean yields greatly decrease and first order risk increases substantially with doubling of CO{sub 2}. The commonly suggested mitigation methods such as adjusting cultivars and planting dates did not alleviate the weather impact and associated risk sufficiently to lead to a productive, alternative cropping system.

  17. Mechanical reliability of double clad fibers in typical fiber laser deployment conditions

    NASA Astrophysics Data System (ADS)

    Walorny, Michael; Abramczyk, Jaroslaw; Jacobson, Nick; Tankala, Kanishka

    2016-03-01

    With the rapid acceptance of fiber lasers and amplifiers for various materials processing and defense applications the long term optical and mechanical reliability of the fiber laser, and therefore the components that make up the laser, is of significant interest to the industrial and defense communities. The double clad fiber used in a fiber laser is a key component whose lifetime in typical deployment conditions needs to be understood. The optical reliability of double clad fiber has recently been studied and a predictive model of fiber lifetime has been published. In contrast, a rigorous model for the mechanical reliability of the fiber and an analysis of the variables affecting the lifetime of the fiber in typical deployment conditions has not been studied. This paper uses the COST-218 model which is widely used for analyzing the mechanical lifetime of fiber used in the telecom industry. The factors affecting lifetime are analyzed to make the reader aware of the design choices a laser manufacturer can make, and the information they must seek from fiber suppliers, to ensure excellent lifetime for double clad fiber and consequently for the fiber laser. It is shown that the fiber's stress corrosion susceptibility, its proof strength, the coil diameter and the length of fiber coiled to achieve good beam quality all have important implications on fiber lifetime.

  18. Conditions for double layers in the earth's magnetosphere and perhaps in other astrophysical objects

    NASA Technical Reports Server (NTRS)

    Lyons, L. R.

    1987-01-01

    It is suggested that the features which govern the formation of the double layers are: (1) the divergence of the magnetospheric electric field, (2) the ionospheric conductivity, and (3) the current-voltage characteristics of auroral magnetic field lines. Also considered are conditions in other astrophysical objects that could lead to the formation of DLs in a manner analogous to what occurs in the earth's auroral zones. It is noted that two processes can drive divergent Pedersen currents within a collisional conducting layer: (1) sheared plasma flow applied anywhere along the magnetic field lines connected to the conducting layer and (2) a neutral flow with shear within the conducting layer.

  19. Two Bessel Bridges Conditioned Never to Collide, Double Dirichlet Series, and Jacobi Theta Function

    NASA Astrophysics Data System (ADS)

    Katori, Makoto; Izumi, Minami; Kobayashi, Naoki

    2008-06-01

    It is known that the moments of the maximum value of a one-dimensional conditional Brownian motion, the three-dimensional Bessel bridge with duration 1 started from the origin, are expressed using the Riemann zeta function. We consider a system of two Bessel bridges, in which noncolliding condition is imposed. We show that the moments of the maximum value is then expressed using the double Dirichlet series, or using the integrals of products of the Jacobi theta functions and its derivatives. Since the present system will be provided as a diffusion scaling limit of a version of vicious walker model, the ensemble of 2-watermelons with a wall, the dominant terms in long-time asymptotics of moments of height of 2-watermelons are completely determined. For the height of 2-watermelons with a wall, the average value was recently studied by Fulmek by a method of enumerative combinatorics.

  20. Mechanisms of Steady and Oscillatory Double-component Convection Due To Different Boundary Conditions

    NASA Astrophysics Data System (ADS)

    Tsitverblit, Naftali

    The effects of different boundary conditions on the behavior of double-component fluid are of basic significance for the ocean processes up to the scales of the global ocean thermohaline circulation [Walin, Palaeogeogr. Palaeoclimatol. Palaeoecol. 50, 323 (1985)]. One major aspect of such flows is that the effect of different boundary conditions can be conceptually analogous to that of different diffusivities in the classi- cal double-diffusion [Welander, Tellus, Ser. A 41, 66 (1989); Tsitverblit, Phys. Fluids 9, 2458 (1997); ibid. 11, 2516 (1999); Phys. Rev. E 62, R7591 (2000)]. This work reports the existence of a novel mechanism of double-component convection resulting from different boundary conditions, whose nature is unrelated to differential diffu- sion. This mechanism emerges in a horizontal layer of Boussinesq fluid as a stable stratification due to flux boundary conditions is added to an unstable gradient speci- fied by fixed boundary values. A large enough perturbation substantially decreases the stable flux gradient but fails to mix the unstable fixed-value gradient. Steady finite- amplitude flows reminiscent of Rayleigh--Benard convection then arise even as the net background stratification is stable. At sufficiently large Rayleigh numbers, con- tinuation of such flows into the finite-amplitude states in Tsitverblit (2000) exhibits a hysteresis region in the slot inclination angle, due to the dissimilar nature of these two convective flows. Welander's (1989) oscillatory instability is also analyzed with the focus on the compensating across-slot gradients. In the inviscid fluid, k = 0 is the most unstable wave number, with the perturbation frequency c = 0. The long- wavelength expansion yields the critical (joint) Rayleigh number, Rac, and group ve- locity, k (c/k), for k = 0 as Rac = 12(k) and k = c c 2 c 60/7. [Generally, at k = 0, the critical (fixed-value) Rayleigh number Rac = (2Ras + 5040)/51 and 12(k) = Ras; Ras being the flux Rayleigh number

  1. Knockout beyond the dripline

    SciTech Connect

    Bonaccorso, A.; Charity, R. J.; Kumar, R.; Salvioni, G.

    2015-02-24

    In this contribution, we will describe neutron and proton removal from {sup 9}C and {sup 7}Be which are two particularly interesting nuclei entering the nucleo-synthesis pp-chain [1, 2]. Neutron and proton removal reactions have been used in the past twenty years to probe the single-particle structure of exotic nuclei. The core parallel-momentum distribution can give information on the angular momentum and spin of the nucleon initial state while the total removal cross section is sensitive to the asymptotic part of the initial wave function and also to the reaction mechanism. Because knockout is a peripheral reaction from which the Asymptotic Normalization Constant (ANC) of the single-particle wave function can be extracted, it has been used as an indirect method to obtain the rate of reactions like {sup 8}B(p,γ){sup 9}C or {sup 7}Be(p,γ){sup 8}B. Nucleon removal has recently been applied by the HiRA collaboration [3] to situations in which the remaining “core” is beyond the drip line, such as {sup 8}C and {sup 6}Be, unbound by one or more protons, and whose excitation-energy spectrum can be obtained by the invariant-mass method. By gating on the ground-state peak, “core” parallel-momentum distributions and total knockout cross sections have been obtained similar to previous studies with well-bound “cores”. In addition for each projectile, knock out to final bound states has also been obtained in several cases. We will report on the theoretical description and comparison to this experimental data for a few cases for which advances in the accuracy of the transfer-to-the continuum model [4, 5] have been made [6]. These include the use, when available, of “ab-initio” overlaps for the initial state [7] and in particular their ANC values [8]. Also, the construction of a nucleus-target folding potential for the treatment of the core-target S-matrix [9] using for the cores “ab-initio” densities [10] and state-of-the-art n−{sup 9}Be optical

  2. Hanford Double-Shell Tank Extent-of-Condition Review - 15498

    SciTech Connect

    Johnson, J. M.; Baide, D. D.; Barnes, T. J.; Boomer, K. D.; Gunter, J. R.; Venetz, T. J.

    2014-11-19

    During routine visual inspections of Hanford double-shell waste tank 241-AY-102 (AY-102), anomalies were identified on the annulus floor which resulted in further evaluations. Following a formal leak assessment in October 2012, Washington River Protection Solutions, LLC (WRPS) determined that the primary tank of AY-102 was leaking. A formal leak assessment, documented in RPP-ASMT-53793, Tank 241-AY-102 Leak Assessment Report, identified first-of-a-kind construction difficulties and trial-and-error repairs as major contributing factors to tank failure.1 To determine if improvements in double-shell tank (DST) construction occurred after construction of tank AY-102, a detailed review and evaluation of historical construction records was performed for Hanford’s remaining twenty-seven DSTs. Review involved research of 241 boxes of historical project documentation to better understand the condition of the Hanford DST farms, noting similarities in construction difficulties/issues to tank AY-102. Information gathered provides valuable insight regarding construction difficulties, future tank operations decisions, and guidance of the current tank inspection program. Should new waste storage tanks be constructed in the future, these reviews also provide valuable lessons-learned.

  3. Hanford Double-Shell Tank Extent-of-Condition Construction Review

    SciTech Connect

    Venetz, Theodore J.; Johnson, Jeremy M.; Gunter, Jason R.; Barnes, Travis J.; Washenfelder, Dennis J.; Boomer, Kayle D.

    2013-11-14

    During routine visual inspections of Hanford double-shell waste tank 241-AY-102 (AY-102), anomalies were identified on the annulus floor which resulted in further evaluations. Following a formal leak assessment in October 2012, Washington River Protection Solutions, LLC (WRPS) determined that the primary tank of AY-102 was leaking. The formal leak assessment, documented in RPP-ASMT-53793,Tank 241-AY-102 Leak Assessment Report, identified first-of-a-kind construction difficulties and trial-and-error repairs as major contributing factors to tank failure. To determine if improvements in double-shell tank (DST) construction occurred after construction of tank AY-102, a detailed review and evaluation of historical construction records were performed for the first three DST tank farms constructed, which included tanks 241-AY-101, 241-AZ-101, 241-AZ-102, 241-SY-101, 241-SY-102, and 241-SY-103. The review for these six tanks involved research and review of dozens of boxes of historical project documentation. These reviews form a basis to better understand the current condition of the three oldest Hanford DST farms. They provide a basis for changes to the current tank inspection program and also provide valuable insight into future tank use decisions. If new tanks are constructed in the future, these reviews provide valuable "lessons-learned" information about expected difficulties as well as construction practices and techniques that are likely to be successful.

  4. Modified and double-clad large mode-area leakage channel fibers for extreme temperature conditions

    NASA Astrophysics Data System (ADS)

    Thavasi Raja, G.; Varshney, Shailendra K.

    2015-03-01

    Recently large-mode-area hybrid leakage channel fibers (HLCFs) were reported to overcome the limitation on mode area with single-mode (SM) operation for the practical bending radius of 7.5 cm at the preferred wavelength of 1064 nm. In this paper, we present the effects of a thermally induced refractive index change on the mode area of bend-compensated extremely LMA modified HLCFs (M-HLCFs) and double-clad M-HLCFs. A full-vectorial finite-element method-based modal solver is used to obtain the modal characteristics of M-HLCFs in various heat load conditions. Numerical simulations reveal that the effective mode area of M-HLCFs is ˜1433 μm2 at room temperature, which marginally decreases to ˜1387 μm2 while SM operation is maintained when the temperature distribution rises to ˜125 °C over the fiber geometry during high-power operations. We have also investigated a double-clad M-HLCF design exhibiting a mode area > ˜ 1000 μm2 for all heat load density variations up to a maximum of 12 × 109 W m-3, corresponding to a 250 °C temperature in the center of the fiber core region.

  5. Gal knockout and beyond.

    PubMed

    Zhong, R

    2007-01-01

    Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted. PMID:17227553

  6. Activation of NADPH-recycling systems in leaves and roots of Arabidopsis thaliana under arsenic-induced stress conditions is accelerated by knock-out of Nudix hydrolase 19 (AtNUDX19) gene.

    PubMed

    Corpas, Francisco J; Aguayo-Trinidad, Simeón; Ogawa, Takahisa; Yoshimura, Kazuya; Shigeoka, Shigeru

    2016-03-15

    NADPH is an important cofactor in cell growth, proliferation and detoxification. Arabidopsis thaliana Nudix hydrolase 19 (AtNUDX19) belongs to a family of proteins defined by the conserved amino-acid sequence GX5-EX7REUXEEXGU which has the capacity to hydrolyze NADPH as a physiological substrate in vivo. Given the importance of NADPH in the cellular redox homeostasis of plants, the present study compares the responses of the main NADPH-recycling systems including NADP-isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and NADP-malic enzyme (ME) in the leaves and roots of Arabidopsis wild-type (Wt) and knock-out (KO) AtNUDX19 mutant (Atnudx19) plants under physiological and arsenic-induced stress conditions. Two major features were observed in the behavior of the main NADPH-recycling systems: (i) under optimal conditions in both organs, the levels of these activities were higher in nudx19 mutants than in Wt plants; and, (ii) under 500μM AsV conditions, these activities increase, especially in nudx19 mutant plants. Moreover, G6PDH activity in roots was the most affected enzyme in both Wt and nudx19 mutant plants, with a 4.6-fold and 5.0-fold increase, respectively. In summary, the data reveals a connection between the absence of chloroplastic AtNUDX19 and the rise in all NADP-dehydrogenase activities under physiological and arsenic-induced stress conditions, particularly in roots. This suggests that AtNUDX19 could be a key factor in modulating the NADPH pool in plants and consequently in redox homeostasis. PMID:26878367

  7. Conditional Knockout of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 in Myeloid Cells Attenuates Renal Fibrosis after Unilateral Ureter Obstruction

    PubMed Central

    Teng, Jing-Fei; Wang, Kai; Li, Yao; Qu, Fa-Jun; Yuan, Qing; Cui, Xin-Gang; Wang, Quan-Xing; Xu, Dan-Feng

    2015-01-01

    Background: Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase. Studies have revealed its roles in various disease, however, whether SHP-2 involves in renal fibrosis remains unclear. The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis. Methods: Myeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system, and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO). The total collagen deposition in the renal interstitium was assessed using picrosirius red stain. F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium. Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney. Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells. Results: Src homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO. Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice. Meanwhile, the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice. However, no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice. Conclusions: Our observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis, and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury. PMID:25947403

  8. Double torsion fracture mechanics testing of shales under chemically reactive conditions

    NASA Astrophysics Data System (ADS)

    Chen, X.; Callahan, O. A.; Holder, J. T.; Olson, J. E.; Eichhubl, P.

    2015-12-01

    Fracture properties of shales is vital for applications such as shale and tight gas development, and seal performance of carbon storage reservoirs. We analyze the fracture behavior from samples of Marcellus, Woodford, and Mancos shales using double-torsion (DT) load relaxation fracture tests. The DT test allows the determination of mode-I fracture toughness (KIC), subcritical crack growth index (SCI), and the stress-intensity factor vs crack velocity (K-V) curves. Samples are tested at ambient air and aqueous conditions with variable ionic concentrations of NaCl and CaCl2, and temperatures up to 70 to determine the effects of chemical/environmental conditions on fracture. Under ambient air condition, KIC determined from DT tests is 1.51±0.32, 0.85±0.25, 1.08±0.17 MPam1/2 for Marcellus, Woodford, and Mancos shales, respectively. Tests under water showed considerable change of KIC compared to ambient condition, with 10.6% increase for Marcellus, 36.5% decrease for Woodford, and 6.7% decrease for Mancos shales. SCI under ambient air condition is between 56 and 80 for the shales tested. The presence of water results in a significant reduction of the SCI from 70% to 85% compared to air condition. Tests under chemically reactive solutions are currently being performed with temperature control. K-V curves under ambient air conditions are linear with stable SCI throughout the load-relaxation period. However, tests conducted under water result in an initial cracking period with SCI values comparable to ambient air tests, which then gradually transition into stable but significantly lower SCI values of 10-20. The non-linear K-V curves reveal that crack propagation in shales is initially limited by the transport of chemical agents due to their low permeability. Only after the initial cracking do interactions at the crack tip lead to cracking controlled by faster stress corrosion reactions. The decrease of SCI in water indicates higher crack propagation velocity due to

  9. IAP gene deletion and conditional knockout models.

    PubMed

    Silke, John; Vaux, David L

    2015-03-01

    Gene deletion studies have helped reveal the unique and overlapping roles played by IAP proteins. Crossing IAP mutant mice has helped unravel the complex feed-back regulatory circuits in which cIAP1, cIAP2 and XIAP allow innate defensive responses to microbial pathogens, without the development of auto-inflammatory syndromes. Deletion of genes for Survivin and its homologs in yeasts, invertebrates and mammals has shown that it functions differently, as it is not a regulator of innate immunity or apoptosis, but acts together with INCENP, aurora kinase B and Borealin to allow chromosome segregation during mitosis. PMID:25545814

  10. De novo inbred heterozygous Zeb2/Sip1 mutant mice uniquely generated by germ-line conditional knockout exhibit craniofacial, callosal and behavioral defects associated with Mowat-Wilson syndrome.

    PubMed

    Takagi, Tsuyoshi; Nishizaki, Yuriko; Matsui, Fumiko; Wakamatsu, Nobuaki; Higashi, Yujiro

    2015-11-15

    Mowat-Wilson syndrome (MOWS) is caused by de novo heterozygous mutation at ZEB2 (SIP1, ZFHX1B) gene, and exhibit moderate to severe intellectual disability (ID), a characteristic facial appearance, epilepsy and other congenital anomalies. Establishing a murine MOWS model is important, not only for investigating the pathogenesis of this disease, but also for identifying compounds that may improve the symptoms. However, because the heterozygous Zeb2 knockout mouse could not be maintained as a mouse line with the inbred C57BL/6 background, it was difficult to use those mice for the study of MOWS. Here, we systematically generated de novo Zeb2 Δex7/+ mice by inducing the Zeb2 mutation in the germ cells using conditional recombination system. The de novo Zeb2 Δex7/+ mice with C57BL/6 background developed multiple defects relevant to MOWS, including craniofacial abnormalities, defective corpus callosum formation and the decreased number of parvalbumin interneurons in the cortex. In behavioral analyses, these mice showed reduced motor activity, increased anxiety and impaired sociability. Notably, during the Barnes maze test, immobile Zeb2 mutant mice were observed over repeated trials. In contrast, neither the mouse line nor the de novo Zeb2 Δex7/+ mice with the closed colony ICR background showed cranial abnormalities or reduced motor activities. These results demonstrate the advantages of using de novo Zeb2 Δex7/+ mice with the C57BL/6 background as the MOWS model. To our knowledge, this is the first time an inducible de novo mutation system has been applied to murine germline cells to produce an animal model of a human congenital disease. PMID:26319231

  11. Ambient-temperature Conditioning as a Probe of Double-C Transformation Mechanisms in Pu-2.0 at. % Ga

    SciTech Connect

    Jeffries, J R; Blobaum, K M; Wall, M A; Schwartz, A J

    2008-04-02

    The gallium-stabilized Pu-2.0 at. % Ga alloy undergoes a partial or incomplete low-temperature martensitic transformation from the metastable {delta} phase to the gallium-containing, monoclinic {alpha}{prime} phase near -100 C. This transformation has been shown to occur isothermally and it displays anomalous double-C kinetics in a time-temperature-transformation (TTT) diagram, where two nose temperatures anchoring an upper- and lower-C describe minima in the time for the initiation of transformation. The underlying mechanisms responsible for the double-C behavior are currently unresolved, although recent experiments suggest that a conditioning treatment--wherein, following an anneal at 375 C, the sample is held at a sub-anneal temperature for a period of time--significantly influences the upper-C of the TTT diagram. As such, elucidating the effects of the conditioning treatment upon the {delta} {yields} {alpha}{prime} transformation can provide valuable insights into the fundamental mechanisms governing the double-C kinetics of the transition. Following a high-temperature anneal, a differential scanning calorimeter (DSC) was used to establish an optimal conditioning curve that depicts the amount of {alpha}{prime} formed during the transformation as a function of conditioning temperature for a specified time. With the optimal conditioning curve as a baseline, the DSC was used to explore the circumstances under which the effects of the conditioning treatment were destroyed, resulting in little or no transformation.

  12. Conditions for double layers in the Earth's magnetosphere and perhaps in other astrophysical objects

    NASA Technical Reports Server (NTRS)

    Lyons, L. R.

    1987-01-01

    Double layers form along auroral field lines in the Earth's magnetosphere. They form in order to maintain current continuity in the ionosphere in the presence of a magnetospheric electric field E with nabla x E is not equal to 0. Features which govern the formation of the double layers are: (1) the divergence of E, (2) the conductivity of the ionosphere, and (3) the current-voltage characteristics of auroral magnetic field lines. Astrophysical situations where nabla x E is not equal to 0 is applied to a conducting plasma similar to the Earth's ionosphere are potential candidates for the formation of double layers. The region with nabla x E is not equal to 0 can be generated within, or along field lines connected to, the conducting plasma. In addition to nabla x E, shear neutral flow in the conducting plasma can also form double layers.

  13. Laboratory studies of kinetic instabilities under double plasma resonance condition in a mirror-confined non-equilibrium plasma

    NASA Astrophysics Data System (ADS)

    Viktorov, Mikhail; Golubev, Sergey; Mansfeld, Dmitry; Vodopyanov, Alexander; Zaitsev, Valery

    2016-04-01

    Plasma instabilities in magnetic traps on the Sun are the sources of powerful broadband radio emission (the so-called type IV bursts) which is interpreted as the excitation of plasma waves by fast electrons in the upper hybrid resonance frequency followed by transformation in electromagnetic waves. In the case of double plasma resonance condition when the frequency of the upper hybrid resonance coincides with one of the electron gyrofrequency harmonics the instability increment of plasma waves is greatly increased. This leads to the appearance of bright narrow-band radio emission near the harmonics of the electron gyrofrequency - the so-called zebra patterns. With the use of non-equilibrium mirror-confined plasma produced by the electron cyclotron resonance (ECR) discharge we provide the possibility to study plasma instabilities under double plasma resonance condition in the laboratory. In the experiment such conditions are fulfilled just after ECR heating switch-off, i.e. in the very beginning of a dense plasma decay phase. The observed instability is accompanied by a pulse-periodic generation of a powerful electromagnetic radiation at a frequency close to the upper hybrid resonance frequency and a second harmonic of the electron gyrofrequency, and synchronous precipitations of fast electrons from the trap ends. It is shown that the observed instability is due to the excitation of plasma waves at a double plasma resonance in decaying plasma of the ECR discharge. Possible manifestations of double plasma resonance effect are not rare in astrophysical plasmas. The phenomenon of zebra pattern is observed not only on the Sun, but in the decametric radiation of the Jupiter, kilometric radiation of the Earth and even in the radio emissions of pulsars. Thus, verification of the effect of double plasma resonance in a laboratory plasma experiments is a very relevant task.

  14. Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice

    PubMed Central

    Belizário, José; Vieira-Cordeiro, Luiz; Enns, Sylvia

    2015-01-01

    Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents) or intracellular (DNA damage and Ca2+ overload) stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER), cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1), RIPK3, and the mixed kinase domain-like (MLKL) for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice. PMID:26491219

  15. Knockout of Extracytoplasmic Function Sigma Factor ECF-10 Affects Stress Resistance and Biofilm Formation in Pseudomonas putida KT2440

    PubMed Central

    Tettmann, Beatrix; Dötsch, Andreas; Armant, Olivier; Fjell, Christopher D.

    2014-01-01

    Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA, ttgB, and ttgC, which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440. PMID:24907323

  16. Designer Nuclease-Mediated Generation of Knockout THP1 Cells.

    PubMed

    Schmidt, Tobias; Schmid-Burgk, Jonathan L; Ebert, Thomas S; Gaidt, Moritz M; Hornung, Veit

    2016-01-01

    Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations. PMID:26443227

  17. Ppp2ca knockout in mice spermatogenesis.

    PubMed

    Pan, Xiaoyun; Chen, Xia; Tong, Xin; Tang, Chao; Li, Jianmin

    2015-04-01

    Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase involved in meiosis, mitosis, sperm capacitation, and apoptosis. Abberant activity of PP2A has been associated with a number of diseases. The homolog PPP2CA and PPP2CB can each function as the phosphatase catalytic subunit generally referred to as PP2AC. We generated a Ppp2ca conditional knockout (CKO) in C57BL/6J mice. Exon 2 of Ppp2ca was knocked out in a spatial or temporal-specific manner in primordial germ cells at E12.5. This Ppp2ca-null mutation caused infertility in male C57BL/6J mice. These CKO mice provide a powerful tool to study the mechanisms of Ppp2ca in development and disease. PMID:25628439

  18. Graphene-like Two-Dimensional Ionic Boron with Double Dirac Cones at Ambient Condition.

    PubMed

    Ma, Fengxian; Jiao, Yalong; Gao, Guoping; Gu, Yuantong; Bilic, Ante; Chen, Zhongfang; Du, Aijun

    2016-05-11

    Recently, partially ionic boron (γ-B28) has been predicted and observed in pure boron, in bulk phase and controlled by pressure [ Nature 2009 , 457 , 863 ]. By using ab initio evolutionary structure search, we report the prediction of ionic boron at a reduced dimension and ambient pressure, namely, the two-dimensional (2D) ionic boron. This 2D boron structure consists of graphene-like plane and B2 atom pairs with the P6/mmm space group and six atoms in the unit cell and has lower energy than the previously reported α-sheet structure and its analogues. Its dynamical and thermal stability are confirmed by the phonon-spectrum and ab initio molecular dynamics simulation. In addition, this phase exhibits double Dirac cones with massless Dirac Fermions due to the significant charge transfer between the graphene-like plane and B2 pair that enhances the energetic stability of the P6/mmm boron. A Fermi velocity (vf) as high as 2.3 × 10(6) m/s, which is even higher than that of graphene (0.82 × 10(6) m/s), is predicted for the P6/mmm boron. The present work is the first report of the 2D ionic boron at atmospheric pressure. The unique electronic structure renders the 2D ionic boron a promising 2D material for applications in nanoelectronics. PMID:27050491

  19. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

    NASA Astrophysics Data System (ADS)

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  20. Comparison between dexmedetomidine and fentanyl on intubation conditions during awake fiberoptic bronchoscopy: A randomized double-blind prospective study

    PubMed Central

    Mondal, Sudeshna; Ghosh, Sarmila; Bhattacharya, Susmita; Choudhury, Brojen; Mallick, Suchismita; Prasad, Anu

    2015-01-01

    Background and Aims: Various drugs are used for providing favorable intubation conditions during awake fiberoptic intubation (AFOI). However, most of them cause respiratory depression and airway obstruction leading to hypoxemia. The aim of this study was to compare intubation conditions, and incidence of desaturation between dexmedetomidine and fentanyl group during AFOI. Material and Methods: This randomized double-blind prospective study was conducted on a total of 60 patients scheduled for elective laparotomies who were randomly allocated into two groups: Group A received dexmedetomidine 1 mcg/kg and Group B received fentanyl 2 mcg/kg over 10 min. Patients in both groups received glycopyrrolate 0.2 mg intravenous, nebulization with 2% lidocaine 4 ml over 20 min and 10% lidocaine spray before undergoing AFOI. Adequacy of intubation condition was evaluated by cough score and post-intubation score. Incidence of desaturation, hemodynamic changes and sedation using Ramsay sedation scale (RSS) were noted and compared between two groups. Results: Cough Score (1-4), post-intubation Score (1-3) and RSS (1-6) were significantly favorable (P < 0.0001) along with minimum hemodynamic responses to intubation (P < 0.05) and less oxygen desaturation (P < 0.0001) in Group A than Group B. Conclusion: Dexmedetomidine is more effective than fentanyl in producing better intubation conditions, sedation along with hemodynamic stability and less desaturation during AFOI. PMID:25948903

  1. A review of current large-scale mouse knockout efforts.

    PubMed

    Guan, Chunmei; Ye, Chao; Yang, Xiaomei; Gao, Jiangang

    2010-02-01

    After the successful completion of the human genome project (HGP), biological research in the postgenome era urgently needs an efficient approach for functional analysis of genes. Utilization of knockout mouse models has been powerful for elucidating the function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and gene targeting are two independent techniques for making knockout mice from embryonic stem (ES) cells. Gene trapping is high-throughput, random, and sequence-tagged while gene targeting enables the knockout of specific genes. It has been about 20 years since the first gene targeting and gene trapping mice were generated. In recent years, new tools have emerged for both gene targeting and gene trapping, and organizations have been formed to knock out genes in the mouse genome using either of the two methods. The knockout mouse project (KOMP) and the international gene trap consortium (IGTC) were initiated to create convenient resources for scientific research worldwide and knock out all the mouse genes. Organizers of KOMP regard it as important as the HGP. Gene targeting methods have changed from conventional gene targeting to high-throughput conditional gene targeting. The combined advantages of trapping and targeting elements are improving the gene trapping spectrum and gene targeting efficiency. As a newly-developed insertional mutation system, transposons have some advantages over retrovirus in trapping genes. Emergence of the international knockout mouse consortium (IKMP) is the beginning of a global collaboration to systematically knock out all the genes in the mouse genome for functional genomic research. PMID:20095055

  2. Marginal and Conditional Distribution Estimation from Double-Sampled Semi-Competing Risks Data

    PubMed Central

    Yu, Menggang; Yiannoutsos, Constantin T

    2015-01-01

    Informative dropout is a vexing problem for any biomedical study. Most existing statistical methods attempt to correct estimation bias related to this phenomenon by specifying unverifiable assumptions about the dropout mechanism. We consider a cohort study in Africa that uses an outreach program to ascertain the vital status for dropout subjects. These data can be used to identify a number of relevant distributions. However, as only a subset of dropout subjects were followed, vital status ascertainment was incomplete. We use semi-competing risk methods as our analysis framework to address this specific case where the terminal event is incompletely ascertained and consider various procedures for estimating the marginal distribution of dropout and the marginal and conditional distributions of survival. We also consider model selection and estimation efficiency in our setting. Performance of the proposed methods is demonstrated via simulations, asymptotic study, and analysis of the study data. PMID:26924877

  3. A Double Blind Randomized Clinical Trial of Remote Ischemic Conditioning in Live Donor Renal Transplantation

    PubMed Central

    Nicholson, Michael L.; Pattenden, Clare J.; Barlow, Adam D.; Hunter, James P.; Lee, Gwyn; Hosgood, Sarah A.

    2015-01-01

    Abstract Ischemic conditioning involves the delivery of short cycles of reversible ischemic injury in order to induce protection against subsequent more prolonged ischemia. This randomized controlled trial was designed to determine the safety and efficacy of remote ischemic conditioning (RC) in live donor kidney transplantation. This prospective randomized clinical trial, 80 patients undergoing live donor kidney transplantation were randomly assigned in a 1:1 ratio to either RC or to a control group. RC consisted of cycles of lower limb ischemia induced by an arterial tourniquet cuff placed around the patient's thigh. In the RC treatment group, the cuff was inflated to 200 mm Hg or systolic pressure +25 mm Hg for 4 cycles of 5 min ischemia followed by 5 min reperfusion. In the control group, the blood pressure cuff was inflated to 25 mm Hg. Patients and medical staff were blinded to treatment allocation. The primary end-point was renal function measured by estimated glomerular filtration rate (eGFR) at 1 and 3 months posttransplant. Donor and recipient demographics were similar in both groups (P < 0.05). There were no significant differences in eGFR at 1 month (control 52 ± 14 vs RC 54 ± 17 mL/min; P = 0.686) or 3 months (control 50 ± 14 vs RC 49 ± 18 mL/min; P = 0.678) between the control and RC treatment groups. The RC technique did not cause any serious adverse effects. RC, using the protocol described here, did not improve renal function after live donor kidney transplantation. PMID:26252316

  4. Conditions for observing emergent SU(4) symmetry in a double quantum dot

    NASA Astrophysics Data System (ADS)

    Nishikawa, Yunori; Curtin, Oliver J.; Hewson, Alex C.; Crow, Daniel J. G.; Bauer, Johannes

    2016-06-01

    We analyze conditions for the observation of a low-energy SU(4) fixed point in capacitively coupled quantum dots. One problem, due to dots with different couplings to their baths, has been considered by L. Tosi, P. Roura-Bas, and A. A. Aligia, J. Phys.: Condens. Matter 27, 335601 (2015), 10.1088/0953-8984/27/33/335601. They showed how symmetry can be effectively restored via the adjustment of individual gates voltages, but they make the assumption of infinite on-dot and interdot interaction strengths. A related problem is the difference in the magnitudes between the on-dot and interdot strengths for capacitively coupled quantum dots. Here we examine both factors, based on a two-site Anderson model, using the numerical renormalization group to calculate the local spectral densities on the dots and the renormalized parameters that specify the low-energy fixed point. Our results support the conclusions of Tosi et al. that low-energy SU(4) symmetry can be restored, but asymptotically achieved only if the interdot interaction U12 is greater than or of the order of the bandwidth of the coupled conduction bath D , which might be difficult to achieve experimentally. By comparing the SU(4) Kondo results for a total dot occupation ntot=1 and 2, we conclude that the temperature dependence of the conductance is largely determined by the constraints of the Friedel sum rule rather than the SU(4) symmetry and suggest that an initial increase of the conductance with temperature is a distinguishing characteristic feature of an ntot=1 universal SU(4) fixed point.

  5. Electric Double Layer Capacitor (EDLC) based Mismatching Losses Reduction under Fast-Shaded Conditions of PV Modules

    NASA Astrophysics Data System (ADS)

    Syafaruddin; Tanaka, Yasuyuki; Karatepe, Engin; Hiyama, Takashi

    Fast-moving irradiance condition is one of problems that need to be solved in the non-stationary conventional maximum power point (MPP) trackers of PV system. Under sudden irradiance changes, the output power is changed drastically that leads to the shifting in MPP voltage. Conventional MPP algorithms may start continuously to search for finding the optimum point. However, suddenly another shadow can occur prior to complete removing of previous shadow. Continuing the tracking process under this condition will cause to lose energy. This paper presents the electric double layer capacitor (EDLC) as the power compensation method for improving the maximum power transfer of PV system under short-term period of shading. Several scenarios are tested in this work by measurement the percentage of power compensation, for instance the effect of capacitor size to the period of shading, the effects of shading period to the level shading intensity and cell temperature. This paper is directly purposed to reduce the power losses for moving objects powered by solar energy, such as solar car and solar boat systems.

  6. Dynamics of electrical double layer formation in room-temperature ionic liquids under constant-current charging conditions

    SciTech Connect

    Jiang, Xikai; Huang, Jingsong; Zhao, Hui; Sumpter, Bobby G; Qiao, Rui

    2014-01-01

    We report detailed simulation results on the formation dynamics of an electrical double layer (EDL) inside an electrochemical cell featuring room-temperature ionic liquids (RTILs) enclosed between two planar electrodes. Under relatively small charging currents, the evolution of cell potential during charging can be suitably predicted by the Landau-Ginzburg-type continuum model proposed recently (M. Z. Bazant, B. D. Storey, and A. A. Kornyshev, Phys. Rev. Lett., 106, 046102, 2011). Under very large charging currents, the cell potential shows pronounced oscillation during the initial stage of charging, a feature not captured by the continuum model. Such oscillation originates from the sequential growth of the ionic space charge layers near the electrode surface, allowing the evolution of EDLs in RTILs with time, an atomistic process difficult to visualize experimentally, to be studied by analyzing the cell potential under constant current charging conditions. While the continuum model cannot predict the potential oscillation under such far-from-equilibrium charging conditions, it can nevertheless qualitatively capture the growth of cell potential during the later stage of charging. Improving the continuum model by introducing frequency-dependent dielectric constant and density-dependent ion diffusion coefficients may help to further extend the applicability of the model. Keywords: ionic

  7. Dynamics of electrical double layer formation in room-temperature ionic liquids under constant-current charging conditions.

    PubMed

    Jiang, Xikai; Huang, Jingsong; Zhao, Hui; Sumpter, Bobby G; Qiao, Rui

    2014-07-16

    We report detailed simulation results on the formation dynamics of an electrical double layer (EDL) inside an electrochemical cell featuring room-temperature ionic liquids (RTILs) enclosed between two planar electrodes. Under relatively small charging currents, the evolution of cell potential from molecular dynamics (MD) simulations during charging can be suitably predicted by the Landau-Ginzburg-type continuum model proposed recently (Bazant et al 2011 Phys. Rev. Lett. 106 046102). Under very large charging currents, the cell potential from MD simulations shows pronounced oscillation during the initial stage of charging, a feature not captured by the continuum model. Such oscillation originates from the sequential growth of the ionic space charge layers near the electrode surface. This allows the evolution of EDLs in RTILs with time, an atomistic process difficult to visualize experimentally, to be studied by analyzing the cell potential under constant-current charging conditions. While the continuum model cannot predict the potential oscillation under such far-from-equilibrium charging conditions, it can nevertheless qualitatively capture the growth of cell potential during the later stage of charging. Improving the continuum model by introducing frequency-dependent dielectric constant and density-dependent ion diffusion coefficients may help to further extend the applicability of the model. The evolution of ion density profiles is also compared between the MD and the continuum model, showing good agreement. PMID:24919471

  8. Merger Rates of Double Neutron Stars and Stellar Origin Black Holes: The Impact of Initial Conditions on Binary Evolution Predictions

    NASA Astrophysics Data System (ADS)

    de Mink, S. E.; Belczynski, K.

    2015-11-01

    The initial mass function (IMF), binary fraction, and distributions of binary parameters (mass ratios, separations, and eccentricities) are indispensable inputs for simulations of stellar populations. It is often claimed that these are poorly constrained, significantly affecting evolutionary predictions. Recently, dedicated observing campaigns have provided new constraints on the initial conditions for massive stars. Findings include a larger close binary fraction and a stronger preference for very tight systems. We investigate the impact on the predicted merger rates of neutron stars and black holes. Despite the changes with previous assumptions, we only find an increase of less than a factor of 2 (insignificant compared with evolutionary uncertainties of typically a factor of 10–100). We further show that the uncertainties in the new initial binary properties do not significantly affect (within a factor of 2) our predictions of double compact object merger rates. An exception is the uncertainty in IMF (variations by a factor of 6 up and down). No significant changes in the distributions of final component masses, mass ratios, chirp masses, and delay times are found. We conclude that the predictions are, for practical purposes, robust against uncertainties in the initial conditions concerning binary parameters, with the exception of the IMF. This eliminates an important layer of the many uncertain assumptions affecting the predictions of merger detection rates with the gravitational wave detectors aLIGO/aVirgo.

  9. Synthesis of [Zn-Al-CO 3] layered double hydroxides by a coprecipitation method under steady-state conditions

    NASA Astrophysics Data System (ADS)

    Chang, Z.; Evans, D. G.; Duan, X.; Vial, C.; Ghanbaja, J.; Prevot, V.; de Roy, M.; Forano, C.

    2005-09-01

    A continuous co-precipitation method under steady-state conditions has been investigated for the preparation of nanometer-size layered double hydroxide (LDH) particles using Zn 2Al(OH) 6(CO 3) 0.5·2H 2O as a prototype. The objective was to shorten the preparation time by working without an aging step, using a short and controlled residence time in order to maintain a constant supersaturation level in the reactor and constant particle properties in the exit stream over time. The effects of varying the operating conditions on the structural and textural properties of the LDHs have been studied, including total cation concentration, solvent, residence time, pH and intercalation anion. The products have been characterized using ICP, XRD, FTIR, BET, SEM and TEM. The LDHs prepared by the continuous coprecipitation method have a poorer crystallinity and lower crystallite sizes than those synthesized by the conventional batch method. The results have shown that increasing either cation concentration or the fraction of monoethylene glycol (MEG) in MEG/H 2O mixtures up to 80% (v/v) affect salt solubility and supersaturation, which gives rise to smaller crystallites, larger surface areas and more amorphous compounds. This increase is however limited by the precipitation of zinc and aluminum hydroxides occurring around a total cation concentration of 3.0×10 -1 M in pure water and 3.0×10 -2 M in H 2O/EtOH mixtures. Crystallite size increases with residence time, suggesting a precipitation process controlled by growth. Finally, the continuous coprecipitation method under steady-state conditions has been shown to be a promising alternative to the traditional coprecipitation technique in either pure water or mixed H 2O/MEG solvents.

  10. Impact of ATG-containing reduced-intensity conditioning after single- or double-unit allogeneic cord blood transplantation.

    PubMed

    Pascal, Laurent; Tucunduva, Luciana; Ruggeri, Annalisa; Blaise, Didier; Ceballos, Patrice; Chevallier, Patrice; Cornelissen, Jan; Maillard, Natacha; Tabrizi, Reza; Petersen, Eefke; Linkesch, Werner; Sengeloev, Henrik; Kenzey, Chantal; Pagliuca, Antonio; Holler, Ernst; Einsele, Hermann; Gluckman, Eliane; Rocha, Vanderson; Yakoub-Agha, Ibrahim

    2015-08-20

    We analyzed 661 adult patients who underwent single-unit (n = 226) or double-unit (n = 435) unrelated cord blood transplantation (UCBT) following a reduced-intensity conditioning (RIC) consisting of low-dose total body irradiation (TBI), cyclophosphamide, and fludarabine (Cy/Flu/TBI200). Eighty-two patients received rabbit antithymocyte globulin (ATG) as part of the conditioning regimen (ATG group), whereas 579 did not (non-ATG group). Median age at UCBT was 54 years, and diagnoses were acute leukemias (51%), myelodysplastic syndrome/myeloproliferative neoplasm (19%), and lymphoproliferative diseases (30%). Forty-four percent of patients were transplanted with advanced disease. All patients received ≥4 antigens HLA-matched UCBT. Median number of collected total nucleated cells was 4.4 × 10(7)/kg. In the ATG group, on 64 evaluable patients, ATG was discontinued 1 (n = 27), 2 (n = 20), or > 2 days before the graft infusion (n = 17). In multivariate analyses, the use of ATG was associated with decreased incidence of acute graft-versus-host disease (hazard ratio [HR], 0.31; 95% confidence interval [CI], 0.17-0.55; P < .0001), higher incidence of nonrelapse mortality (HR, 1.68; 95% CI, 1.16-2.43; P = .0009), and decreased overall survival (HR, 1.69; 95% CI, 1.19-2.415; P = .003). Collectively, our results suggest that the use of ATG could be detrimental, especially if given too close to graft infusion in adults undergoing UCBT following Cy/Flu/TBI200 regimen. PMID:26160301

  11. Crack growth behavior under creep-fatigue conditions using compact and double edge notch tension-compression specimens

    NASA Astrophysics Data System (ADS)

    Narasimha Chary, Santosh Balaji

    The American Society for Testing and Materials (ASTM) has recently developed a new standard for creep-fatigue crack growth testing, E 2760-10, that supports testing compact specimens, C(T), under load controlled conditions. C(T) specimens are commonly used for fatigue and creep-fatigue crack growth testing under constant-load-amplitude conditions. The use of these specimens is limited to positive load ratios. They are also limited in the amount of crack growth data that can be developed at high stress intensity values due to accumulation of plastic and/or creep strains leading to ratcheting in the specimen. Testing under displacement control can potentially address these shortcomings of the load-controlled tests for which the C(T) geometry is unsuitable. A double edge notch tension-compression, DEN(T-C), specimen to perform displacement controlled creep-fatigue crack growth testing is developed and optimized with the help of finite element and boundary element analyses. Accurate expressions for estimating the fracture mechanics crack tip parameters such as the stress intensity parameter, K, the crack mouth opening displacement (CMOD), and the load-line displacement (LLD) are developed over a wide range of crack sizes for the DEN(T-C) specimen. A new compliance relationship for use in experimental testing has been developed by using the compliance form available in ASTM E-647 standard. Experimentally determined compliance value compared well with the new relation for C15 steel (AISI 1015) and P91 steel tested at room and elevated temperature conditions respectively. Fatigue crack growth rate data generated using the DEN(T-C) specimens on the two metallic materials are in good agreement with the data generated using standard compact specimens; thus validating the stress-intensity factor and the compliance equation for the double edge notch tension-compression specimen. The testing has shown that the DEN(T-C) specimen is prone to crack asymmetry issues. Through

  12. Effects of D1 receptor knockout on fear and reward learning.

    PubMed

    Abraham, Antony D; Neve, Kim A; Lattal, K Matthew

    2016-09-01

    Dopamine signaling is involved in a variety of neurobiological processes that contribute to learning and memory. D1-like dopamine receptors (including D1 and D5 receptors) are thought to be involved in memory and reward processes, but pharmacological approaches have been limited in their ability to distinguish between D1 and D5 receptors. Here, we examine the effects of a specific knockout of D1 receptors in associative learning tasks involving aversive (shock) or appetitive (cocaine) unconditioned stimuli. We find that D1 knockout mice show similar levels of cued and contextual fear conditioning to WT controls following conditioning protocols involving one, two, or four shocks. D1 knockout mice show increased generalization of fear conditioning and extinction across contexts, revealed as increased freezing to a novel context following conditioning and decreased freezing to an extinguished cue during a contextual renewal test. Further, D1 knockout mice show mild enhancements in extinction following an injection of SKF81297, a D1/D5 receptor agonist, suggesting a role for D5 receptors in extinction enhancements induced by nonspecific pharmacological agonists. Finally, although D1 knockout mice show decreased locomotion induced by cocaine, they are able to form a cocaine-induced conditioned place preference. We discuss these findings in terms of the role of dopamine D1 receptors in general learning and memory processes. PMID:27423521

  13. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

    PubMed Central

    Niksch, Paul D.; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, David P.

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  14. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice.

    PubMed

    Wu, Xudong; Indzhykulian, Artur A; Niksch, Paul D; Webber, Roxanna M; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A; Corey, David P

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  15. Double-component convection due to different boundary conditions in an infinite slot diversely oriented to the gravity

    NASA Astrophysics Data System (ADS)

    Tsitverblit, N.

    2007-08-01

    Onset of small-amplitude oscillatory and both small- and finite-amplitude steady double-component convection arising due to component different boundary conditions in an infinite slot is studied for various slot orientations to the gravity. The main focus is on two compensating background gradients of the components. The physical mechanisms underlying steady and oscillatory convection are analyzed from the perspective of a universally consistent understanding of the effects of different boundary conditions. In a horizontal slot with inviscid fluid addressed by Welander [P. Welander, Tellus Ser. A 41 (1989) 66], oscillatory convection sets in with the most unstable wave number and oscillation frequency being zero. Exact expressions for the critical fixed-value background gradient and the respective group velocity at zero wave number are derived from the long-wavelength expansion both for the horizontal slot with independently varying background gradients and for the inclined slot with the compensating gradients. In the horizontal slot with viscous fluid, the dissipation of along-slot perturbation-cell motion reduces efficiency of the oscillatory instability feedback and thus prevents the most unstable wavelength from being infinite. Based on this interpretation, the oscillatory instability of a three-dimensional (3D) nature is predicted for an interval of long two-dimensional (2D) wavelengths in an inclined slot, and such 3D instability is indeed shown to arise. Related general conditions for three-dimensionality of most unstable disturbances are also formulated. As the slot orientation changes from the horizontal by angle θ (⩾π/2), the oscillatory 2D marginal-stability boundaries in inviscid and viscous fluid are expected to eventually transform into respective steady ones. Oscillatory instability in the vertical slot with viscous fluid, first reported by Tsitverblit [N. Tsitverblit, Phys. Rev. E 62 (2000) R7591], is of a quasi-steady nature. Its (new

  16. Double-component convection due to different boundary conditions in an infinite slot diversely oriented to the gravity

    SciTech Connect

    Tsitverblit, N. . E-mail: naftali@eng.tau.ac.il

    2007-08-15

    Onset of small-amplitude oscillatory and both small- and finite-amplitude steady double-component convection arising due to component different boundary conditions in an infinite slot is studied for various slot orientations to the gravity. The main focus is on two compensating background gradients of the components. The physical mechanisms underlying steady and oscillatory convection are analyzed from the perspective of a universally consistent understanding of the effects of different boundary conditions. In a horizontal slot with inviscid fluid addressed by Welander [P. Welander, Tellus Ser. A 41 (1989) 66], oscillatory convection sets in with the most unstable wave number and oscillation frequency being zero. Exact expressions for the critical fixed-value background gradient and the respective group velocity at zero wave number are derived from the long-wavelength expansion both for the horizontal slot with independently varying background gradients and for the inclined slot with the compensating gradients. In the horizontal slot with viscous fluid, the dissipation of along-slot perturbation-cell motion reduces efficiency of the oscillatory instability feedback and thus prevents the most unstable wavelength from being infinite. Based on this interpretation, the oscillatory instability of a three-dimensional (3D) nature is predicted for an interval of long two-dimensional (2D) wavelengths in an inclined slot, and such 3D instability is indeed shown to arise. Related general conditions for three-dimensionality of most unstable disturbances are also formulated. As the slot orientation changes from the horizontal by angle {theta} ({>=}{pi}/2), the oscillatory 2D marginal-stability boundaries in inviscid and viscous fluid are expected to eventually transform into respective steady ones. Oscillatory instability in the vertical slot with viscous fluid, first reported by Tsitverblit [N. Tsitverblit, Phys. Rev. E 62 (2000) R7591], is of a quasi-steady nature. Its (new

  17. Conditional cash transfers and the double burden of malnutrition among children in Colombia: a quasi-experimental study.

    PubMed

    Lopez-Arana, Sandra; Avendano, Mauricio; Forde, Ian; van Lenthe, Frank J; Burdorf, Alex

    2016-05-28

    Conditional cash-transfer (CCT) programmes have been shown to improve the nutritional and health status of children from poor families. However, CCT programmes may have unintended and not fully known consequences by increasing the risk of overweight and obesity. We examined the impact of Familias en Acción (FA), a large CCT programme in Colombia, on the double burden of malnutrition among pre-school and school-aged children. Height and weight were measured before programme enrolment and during follow-ups in 1290 children from thirty-one treatment municipalities, being compared with 1584 children from sixty-two matched control municipalities. We used a difference-in-differences approach to evaluate the effect of FA on children's stunting, BMI z-scores, thinness, overweight and obesity, controlling for individual and municipality-level confounders. At baseline, the prevalences of stunting and overweight were 30·3 and 15·4 %, respectively, in treatment municipalities and 27·9 and 17·4 % in control municipalities. FA was associated with reduced odds of thinness (OR 0·26; 95 % CI 0·09, 0·75) and higher BMI-for-age z-scores (BMI z-scores) (β 0·14; 95 % CI 0·00, 0·27; P<0·05), although the latter was of small clinical significance. The prevalence of stunting, overweight and obesity decreased over time, but the effect of FA on these outcomes was not significant. The CCT programme in Colombia reduced the odds of thinness, but had no effect on stunting, a more prevalent outcome. The FA programme had no effect on overweight or obesity, although BMI z-scores were higher for children under treatment, raising the possibility of an increase of small clinical significance on BMI among pre-school and school-aged children. PMID:26988836

  18. Proton Knock-Out in Hall A

    SciTech Connect

    Kees de Jager

    2002-06-01

    Proton knock-out is studied in a broad program in Hall A at Jefferson Lab. The first experiment performed in Hall A studied the {sup 16}O(e,e'p) reaction. Since then proton knock-out experiments have studied a variety of aspects of that reaction, from single-nucleon properties to its mechanism, such as final-state interactions and two-body currents, in nuclei from {sup 2}H to {sup 16}O. In this review the results of this program will be summarized and an outlook given of future accomplishments.

  19. A Deep Quench Approach to the Optimal Control of an Allen–Cahn Equation with Dynamic Boundary Conditions and Double Obstacles

    SciTech Connect

    Colli, Pierluigi; Farshbaf-Shaker, M. Hassan Sprekels, Jürgen

    2015-02-15

    In this paper, we investigate optimal control problems for Allen-Cahn variational inequalities with a dynamic boundary condition involving double obstacle potentials and the Laplace-Beltrami operator. The approach covers both the cases of distributed controls and of boundary controls. The cost functional is of standard tracking type, and box constraints for the controls are prescribed. We prove existence of optimal controls and derive first-order necessary conditions of optimality. The general strategy is the following: we use the results that were recently established by two of the authors for the case of (differentiable) logarithmic potentials and perform a so-called “deep quench limit”. Using compactness and monotonicity arguments, it is shown that this strategy leads to the desired first-order necessary optimality conditions for the case of (non-differentiable) double obstacle potentials.

  20. Comparison of nociceptive behavior in prostaglandin E, F, D, prostacyclin and thromboxane receptor knockout mice.

    PubMed

    Popp, Laura; Häussler, Annett; Olliges, Anke; Nüsing, Rolf; Narumiya, Shuh; Geisslinger, Gerd; Tegeder, Irmgard

    2009-08-01

    Antagonist at specific prostaglandin receptors might provide analgesia with a more favourable toxicity profile compared with cyclooxygenase inhibitors. We analyzed nociceptive responses in prostaglandin D, E, F, prostacyclin and thromboxane receptor knockout mice and mice deficient of cyclooxygenase 1 or 2 to evaluate the contribution of individual prostaglandin receptors for heat, mechanical and formalin-evoked pain. None of the knockouts was uniformly protected from all of these pain stimuli but COX-1 and EP4 receptor knockouts presented with reduced heat pain and EP3 receptor and COX-2 knockout mice had reduced licking responses in the 2nd phase of the formalin assay. This was accompanied with reduced c-Fos immunoreactivity in the spinal cord dorsal horn in EP3 knockouts. Oppositely, heat pain sensitivity was increased in FP, EP1 and EP1+3 double mutant mice possibly due to a loss of FP or EP1 receptor mediated central control of thermal pain sensitivity. Deficiency of either EP2 or DP1 was associated with increased formalin-evoked flinching responses and c-Fos IR in dorsal horn neurons suggesting facilitated spinal cord pain reflex circuity. Thromboxane and prostacyclin receptor knockout mice showed normal pain behavior in all tests. The results suggest a differential, pain-stimulus and site-specific contribution of specific PG-receptors for the processing of the nociceptive stimuli, a differential modulation of nociceptive responses by COX-1 and COX-2 derived prostaglandins and compensatory and/or developmental adaptations in mice lacking specific PG receptors. PMID:18938093

  1. Quantifying the role of immobile water on pollutant fluxes in double-permeable media under dynamic flow conditions

    NASA Astrophysics Data System (ADS)

    Knorr, Bastian; Krämer, Florian; Stumpp, Christine; Maloszewski, Piotr

    2014-05-01

    Sustainable use of water resources and their protection against pollution requires fundamental understanding of filter, buffer and storage functions of groundwater systems. Of particular importance are heterogeneous porous aquifers including zones with mobile and immobile water. Pollutants diffuse from high permeable areas into immobile zones with low permeability. Consequently, pollutants can be stored in such immobile water regions and their residence time in double-permeable aquifers is much longer compared to water residence times. However, it still remains unknown how the heterogeneity of an aquifer and time-dependent variability of the water flow influences the pollutant fate in such systems. The objective of this study was to develop experimental and mathematical methods to understand the role of immobile water zones on the pollutant retention, kinetic ad-/desorption and degradation. In saturated column experiments at three different flow rates multitracer experiments were conducted and 4-Chloronitrobenzene (intermediate in the production of explosives) was used as pollutant. The columns were packed with an outer cylinder of clay containing mainly immobile water whereas the centre was filled with coarse quartz sand containing mobile water. In the resulting breakthrough curves of the conservative tracers characterized by different diffusion properties, differences were observed in peak concentration and tailing. These differences indicated a mass exchange with immobile water zones driven by diffusion and were depended on the tracers' molecular diffusion coefficient. The mass exchange increased with decreasing flow rates and was quantified for conservative tracers applying a Single-Fissure Dispersion Model (SFDM) to porous media for the first time. The observed concentrations of the reactive solute 4-Chloronitronbenzen indicated that sorption onto clay minerals enhanced the mass exchange into the immobile water zone. On the other hand sorption and degradation

  2. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

    PubMed Central

    Nakagawa, Yoshiko; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-01-01

    ABSTRACT Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. PMID:27387532

  3. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

    PubMed

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-01-01

    Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. PMID:27387532

  4. Search for optimal conditions for exploring double-parton scattering in four-jet production: kT -factorization approach

    NASA Astrophysics Data System (ADS)

    Kutak, Krzysztof; Maciuła, Rafał; Serino, Mirko; Szczurek, Antoni; van Hameren, Andreas

    2016-07-01

    In the present paper, we discuss how to maximize the double-parton scattering (DPS) contribution in four-jet production by selecting kinematical cuts. Here both single-parton and double-parton scattering effects are calculated in the kT -factorization approach, following our recent developments of relevant methods and tools. Several differential distributions are shown and discussed in the context of future searches for DPS effects, such as rapidity of jets, rapidity distance, and azimuthal correlations between jets. The dependence of the relative DPS amount is studied as a function of those observables. The regions with an enhanced DPS contribution are identified. Future experimental explorations could extract more precise values of σeff and its potential dependence on kinematical variables.

  5. Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice

    PubMed Central

    White, Carl W.; Choong, Yan-Ting; Short, Jennifer L.; Exintaris, Betty; Malone, Daniel T.; Allen, Andrew M.; Evans, Richard J.; Ventura, Sabatino

    2013-01-01

    Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive. PMID:24297884

  6. Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice.

    PubMed

    White, Carl W; Choong, Yan-Ting; Short, Jennifer L; Exintaris, Betty; Malone, Daniel T; Allen, Andrew M; Evans, Richard J; Ventura, Sabatino

    2013-12-17

    Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive. PMID:24297884

  7. Glycogen storage disease type III: A novel Agl knockout mouse model.

    PubMed

    Pagliarani, Serena; Lucchiari, Sabrina; Ulzi, Gianna; Violano, Raffaella; Ripolone, Michela; Bordoni, Andreina; Nizzardo, Monica; Gatti, Stefano; Corti, Stefania; Moggio, Maurizio; Bresolin, Nereo; Comi, Giacomo P

    2014-11-01

    Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease. PMID:25092169

  8. 8He cluster structure studied by recoil proton tagged knockout reaction

    NASA Astrophysics Data System (ADS)

    Ye, Y.; Cao, Z.; Xiao, J.; Jiang, D.; Zheng, T.; Hua, H.; Ge, Y.; Li, X.; Lou, J.; Li, Q.; Lv, L.; Qiao, R.; You, H.; Chen, R.; Sakurai, H.; Otsu, H.; Li, Z.; Nishimura, M.; Sakaguchi, S.; Baba, H.; Togano, Y.; Yoneda, K.; Li, C.; Wang, S.; Wang, H.; Li, K.; Nakayama, Y.; Kondo, Y.; Deguchi, S.; Sato, Y.; Tshoo, K.

    2013-04-01

    Knockout reaction experiment for 8He at 82.3 MeV/u on Hydrogen target was carried out at the RIPS beam line in RIKEN. Recoil protons were detected in coincidence with the forward moving core fragments and neutrons. The quasi-free knockout mechanism is identified through the polar angle correlation and checked by various kinematics conditions. The absolute differential cross sections for 6He core cluster are obtained and compared with the simple Glauber model calculations. The extracted spectroscopic factor is close to unity and a shrinking of the cluster size is evidenced.

  9. Functional consequences of hippocampal neuronal ectopia in the apolipoprotein E receptor-2 knockout mouse

    PubMed Central

    Fish, Kenneth. N.; Krucker, Thomas

    2008-01-01

    Little is known about the impact ectopically located neurons have on the functional connectivity of local circuits. The ApoER2 knockout mouse has subtle cytoarchitectural disruptions, altered prepulse inhibition, and memory abnormalities. We evaluated this mouse mutant as a model to study the role ectopic neurons play in the manifestation of symptoms associated with brain diseases. We found that ectopic CA1 pyramidal and inhibitory neurons in the ApoER2 knockout hippocampus are organized into two distinct stratum pyramidale layers. In vitro analyses found that ApoER2 is not required for neurons to reach maturity in regards to dendritic arborization and synaptic structure density, and electrophysiological testing determined that neurons in both strata pyramidale are integrated into the hippocampal network. However, the presence of these two layers alters the spatiotemporal pattern of hippocampal activity, which may explain why ApoER2 knockout mice have selective cognitive dysfunctions that are revealed only under challenging conditions. PMID:18778775

  10. BDNF restricted knockout mice as an animal model for aggression

    PubMed Central

    Ito, Wataru; Chehab, Mahmoud; Thakur, Siddarth; Li, Jiayang; Morozov, Alexei

    2011-01-01

    Mice with global deletion of one BDNF allele, or with forebrain-restricted deletion of both alleles show elevated aggression, but this phenotype is accompanied by other behavioral changes, including increases in anxiety and deficits in cognition. Here, we performed behavioral characterization of conditional BDNF knockout mice generated using a Cre recombinase driver line, KA1-Cre, which expresses Cre in few areas of brain: highly at hippocampal area CA3, moderately in dentate gyrus, cerebellum and facial nerve nucleus. The mutant animals exhibited elevated conspecific aggression and social dominance, but did not show changes in anxiety-like behaviors assessed using the elevated plus maze and open field test. There were no changes in depression like behaviors tested in the forced swim test, but small increase in immobility in the tail suspension test. In cognitive tasks, mutants showed normal social recognition and normal spatial and fear memory, but exhibited a deficit in object recognition. Thus, this knockout can serve as a robust model of BDNF-dependent aggression and object recognition deficiency. PMID:21255268

  11. Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates by a phaZ-knockout strain of Pseudomonas putida KT2440.

    PubMed

    Vo, Minh Tri; Ko, Kenton; Ramsay, Bruce

    2015-04-01

    A medium-chain-length poly-3-hydroxyalkanote (MCL-PHA) depolymerase knockout mutant of Pseudomonas putida KT2440 was produced by double homologous recombination. A carbon-limited shake-flask study confirmed that depolymerase activity was eliminated. Lysis of both mutant and wild-type strains occurred under these conditions. In carbon-limited, fed-batch culture, the yield of unsaturated monomers from unsaturated substrate averaged only 0.62 mol mol(-1) for the phaZ minus strain compared to 0.72 mol mol(-1) for the wild type. The mutant strain also produced more CO2 and less residual biomass from the same amount of carbon substrate. However, most results indicated that elimination of PHA depolymerase activity had little impact on the overall yield of biomass and PHA. PMID:25563970

  12. Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

    PubMed

    Tamae, Cindy; Liu, Anne; Kim, Katherine; Sitz, Daniel; Hong, Jeeyoon; Becket, Elinne; Bui, Ann; Solaimani, Parrisa; Tran, Katherine P; Yang, Hanjing; Miller, Jeffrey H

    2008-09-01

    We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics. PMID:18621901

  13. An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

    PubMed Central

    Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

    2013-01-01

    SUMMARY PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors. PMID:23471917

  14. Behavior training reverses asymmetry in hippocampal transcriptome of the cav3.2 knockout mice.

    PubMed

    Chung, Ni-Chun; Huang, Ying-Hsueh; Chang, Chuan-Hsiung; Liao, James C; Yang, Chih-Hsien; Chen, Chien-Chang; Liu, Ingrid Y

    2015-01-01

    Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome. PMID:25768289

  15. Characterization of Kidney and Skeleton Phenotypes of Mice Double Heterozygous for Foxc1 and Foxc2.

    PubMed

    Motojima, Masaru; Tanimoto, Sho; Ohtsuka, Masato; Matsusaka, Taiji; Kume, Tsutomu; Abe, Koichiro

    2016-01-01

    Foxc1 and Foxc2 play key roles in mouse development. Foxc1 mutant mice develop duplex kidneys with double ureters, and lack calvarial and sternal bones. Foxc2 null mice have been reported to have glomerular abnormalities in the kidney and axial skeletal anomalies. Expression patterns of Foxc1 and Foxc2 overlap extensively and are believed to have interactive roles. However, cooperative roles of these factors in glomerular and skeletal development are unknown. Therefore, we examined the kidneys and skeleton of mice that were double heterozygous for Foxc1 and Foxc2. Double heterozygotes were generated by mating single heterozygotes for Foxc1 and Foxc2. Newborn double heterozygous mice showed many anomalies in the kidney and urinary tract resembling Foxc1 phenotypes, including duplex kidneys, double ureters, hydronephrosis and mega-ureter. Some mice had hydronephrosis alone. In addition to these macroscopic anomalies, some mice had abnormal glomeruli and disorganized glomerular capillaries observed in Foxc2 phenotypes. Interestingly, these mice also showed glomerular cysts not observed in the single-gene knockout of either Foxc1 or Foxc2 but observed in conditional knockout of Foxc2 in the kidney. Serial section analysis revealed that all cystic glomeruli were connected to proximal tubules, precluding the possibility of atubular glomeruli resulting in cyst formation. Dorsally opened vertebral arches and malformations of sternal bones in the double heterozygotes were phenotypes similar to Foxc1 null mice. Absent or split vertebral bodies in the double heterozygotes were phenotypes similar to Foxc2 null mice, whilst hydrocephalus noted in the Foxc1 phenotype was not observed. Thus, Foxc1 and Foxc2 have a role in kidney and axial skeleton development. These transcription factors might interact in the regulation of the embryogenesis of these organs. PMID:27193493

  16. Clues to VIP function from knockout mice.

    PubMed

    Hamidi, S A; Szema, A M; Lyubsky, S; Dickman, K G; Degene, A; Mathew, S M; Waschek, J A; Said, S I

    2006-07-01

    We have taken advantage of the availability of vasoactive intestinal polypeptide (VIP) knockout (KO) mice to examine the possible influence of deletion of the VIP gene on: (a) airway reactivity and airway inflammation, as indicators of bronchial asthma; (b) mortality from endotoxemia, a model of septic shock; and (c) the pulmonary circulation. VIP KO mice showed: (a) airway hyperresponsiveness to the cholinergic agonist methacholine, as well as peribronchial and perivascular inflammation; (b) a greater susceptibility to death from endotoxemia; and (c) evidence suggestive of pulmonary hypertension. PMID:16888146

  17. Universal statistics of the knockout tournament

    NASA Astrophysics Data System (ADS)

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-11-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness.

  18. Germ line knockout of IGFBP-3 reveals influences of the gene on mammary gland neoplasia.

    PubMed

    Blouin, Marie-José; Bazile, Miguel; Birman, Elena; Zakikhani, Mahvash; Florianova, Livia; Aleynikova, Olga; Powell, David R; Pollak, Michael

    2015-02-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is an important carrier protein for insulin-like growth factors (IGFs) in the circulation. IGFBP-3 antagonizes the growth-promoting and anti-apoptotic activities of IGFs in experimental systems, but in certain contexts can increase IGF bioactivity, probably by increasing its half-life. The goal of this study was to investigate the role of IGFBP-3 in breast carcinogenesis and breast cancer metastasis. In the first part of the study, we exposed IGFBP-3 knockout and wild-type female mice to dimethylbenz[a]anthracene (DMBA) and followed them for appearance of primary tumors for up to 13 months. In the second part, mice of each genotype received an IV injection of 4T1 mammary carcinoma cells and then lung nodules were counted. Our results show that IGFBP-3 knockout mice developed breast tumors significantly earlier than the wild-type (13.9 ± 1.1 versus 22.5 ± 3.3 weeks, respectively, P = 0.0144), suggesting tumor suppression activity of IGFBP-3. In tumors of IGFBP-3 knockout mice, levels of phospho-AKT(Ser473) were increased compared to wild-type mice. The lung metastasis assay showed significantly more and larger lung nodules in IGFBP-3 knockout mice than in wild-type mice. While we observed increased levels of IGFBP-5 protein in the IGFBP-3 knockout mice, our findings suggest that this was not sufficient to completely compensate for the absence of IGFBP-3. Even though knockout of IGFBP-3 is associated with only a subtle phenotype under control conditions, our results reveal that loss of this gene has measurable effects on breast carcinogenesis and breast cancer metastasis. PMID:25614235

  19. Altered Reward Circuitry in the Norepinephrine Transporter Knockout Mouse

    PubMed Central

    Hall, F. Scott; Uhl, George R.; Bearer, Elaine L.; Jacobs, Russell E.

    2013-01-01

    Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn2+ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of

  20. Neuregulin 3 Knockout Mice Exhibit Behaviors Consistent with Psychotic Disorders.

    PubMed

    Hayes, Lindsay N; Shevelkin, Alexey; Zeledon, Mariela; Steel, Gary; Chen, Pei-Lung; Obie, Cassandra; Pulver, Ann; Avramopoulos, Dimitrios; Valle, David; Sawa, Akira; Pletnikov, Mikhail V

    2016-07-01

    Neuregulin 3 (NRG3) is a paralog of NRG1. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, and several intronic single nucleotide polymorphisms in NRG3 are associated with delusions in patients with schizophrenia. In order to gain insights into the biological function of the gene, we generated a novel Nrg3 knockout (KO) mouse model and tested for neurobehavioral phenotypes relevant to psychotic disorders. KO mice displayed novelty-induced hyperactivity, impaired prepulse inhibition of the acoustic startle response, and deficient fear conditioning. No gross cytoarchitectonic or layer abnormalities were noted in the brain of KO mice. Our findings suggest that deletion of the Nrg3 gene leads to alterations consistent with aspects of schizophrenia. We propose that KO mice will provide a valuable animal model to determine the role of the NRG3 in the molecular pathogenesis of schizophrenia and other psychotic disorders. PMID:27606322

  1. Rapid induction of single donor chimerism after double umbilical cord blood transplantation preceded by reduced intensity conditioning: results of the HOVON 106 phase II study

    PubMed Central

    Somers, Judith A.E.; Braakman, Eric; van der Holt, Bronno; Petersen, Eefke J.; Marijt, Erik W.A.; Huisman, Cynthia; Sintnicolaas, Kees; Oudshoorn, Machteld; Groenendijk-Sijnke, Marlies E.; Brand, Anneke; Cornelissen, Jan J.

    2014-01-01

    Double umbilical cord blood transplantation is increasingly applied in the treatment of adult patients with high-risk hematological malignancies and has been associated with improved engraftment as compared to that provided by single unit cord blood transplantation. The mechanism of improved engraftment is, however, still incompletely understood as only one unit survives. In this multicenter phase II study we evaluated engraftment, early chimerism, recovery of different cell lineages and transplant outcome in 53 patients who underwent double cord blood transplantation preceded by a reduced intensity conditioning regimen. Primary graft failure occurred in one patient. Engraftment was observed in 92% of patients with a median time to neutrophil recovery of 36 days (range, 15–102). Ultimate single donor chimerism was established in 94% of patients. Unit predominance occurred by day 11 after transplantation and early CD4+ T-cell chimerism predicted for unit survival. Total nucleated cell viability was also associated with unit survival. With a median follow up of 35 months (range, 10–51), the cumulative incidence of relapse and non-relapse mortality rate at 2 years were 39% and 19%, respectively. Progressionfree survival and overall survival rates at 2 years were 42% (95% confidence interval, 28–56) and 57% (95% confidence interval, 43–70), respectively. Double umbilical cord blood transplantation preceded by a reduced intensity conditioning regimen using cyclophosphamide/fludarabine/4 Gy total body irradiation results in a high engraftment rate with low non-relapse mortality. Moreover, prediction of unit survival by early CD4+ lymphocyte chimerism might suggest a role for CD4+ lymphocyte mediated unit-versus-unit alloreactivity. www.trialregister.nl NTR1573. PMID:25107890

  2. Proteomic Analysis of Loricrin Knockout Mouse Epidermis.

    PubMed

    Rice, Robert H; Durbin-Johnson, Blythe P; Ishitsuka, Yosuke; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Roop, Dennis R

    2016-08-01

    The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates. PMID:27418529

  3. The butyrylcholinesterase knockout mouse a research tool in the study of drug sensitivity, bio-distribution, obesity and Alzheimer's disease.

    PubMed

    Duysen, Ellen G; Li, Bin; Lockridge, Oksana

    2009-05-01

    Butyrylcholinesterase (BChE) mutations common in the human population may result in complete or partial BChE deficiency, making the BChE knockout (KO) mouse a model for human deficiencies. The BChE KO mouse cannot tolerate standard doses of the muscle relaxant succinylcholine or the Alzheimer's disease drugs huperzine A and donepezil. It is resistant to the asthma drug bambuterol. The importance of BChE in detoxication of cocaine has been demonstrated by hepatotoxicity and cardiotoxicity in cocaine-challenged BChE KO mice. The BChE KO mouse becomes obese on a high-fat diet, suggesting a role for BChE in fat metabolism. BChE serves as a backup for acetylcholinesterase by hydrolyzing the neurotransmitter acetylcholine in acetylcholinesterase knockout mice. Imaging studies show that BChE injected intrathecally crosses the blood-brain barrier. Mice, but not humans, have carboxylesterase in their blood. Carboxylesterase obscures the role of BChE in detoxication of organophosphorus pesticides. Future studies will make a double knockout that has neither BChE nor carboxylesterase. The double knockout is expected to be unusually sensitive to the toxicity of organophosphorus pesticides. Knowledge of drug sensitivities in the mouse model of human BChE deficiency will aid in understanding adverse drug effects in humans. PMID:19416087

  4. Fatty acid composition in double and multilayered microcapsules of ω-3 as affected by storage conditions and type of emulsions.

    PubMed

    Jiménez-Martín, Estefanía; Antequera Rojas, Teresa; Gharsallaoui, Adem; Ruiz Carrascal, Jorge; Pérez-Palacios, Trinidad

    2016-03-01

    Spray-dried microcapsules from double (DM) and multilayered (MM) fish oil emulsions were produced to evaluate the effect of type of emulsion on the fatty acid composition during the microencapsulation process and after one month of storage at refrigeration (4°C) and room (20°C) temperature. Encapsulation efficiency, loading and loading efficiency were significantly higher in MM than in DM. C20:5 n-3 (EPA) and C22:6 n-3 (DHA) showed higher proportions in MM than in DM. Some differences in microstructural features were detected, with DM showing cracks and pores. The influence of the storage was significant, decreasing the content of polyunsaturated fatty acids in both MM and DM, above all at 20°C. This decrease was more notable in DM. Multilayered emulsions are more suitable to encapsulate fish oil in terms of quantity of encapsulated oil, microstructure of the microcapsules and protection of fatty acids, especially EPA and DHA, during storage. PMID:26471582

  5. A NEW MODEL FOR MIXING BY DOUBLE-DIFFUSIVE CONVECTION (SEMI-CONVECTION). I. THE CONDITIONS FOR LAYER FORMATION

    SciTech Connect

    Mirouh, G. M.; Garaud, P.; Traxler, A. L.; Wood, T. S.; Stellmach, S.

    2012-05-01

    The process referred to as 'semi-convection' in astrophysics and 'double-diffusive convection in the diffusive regime' in Earth and planetary sciences occurs in stellar and planetary interiors in regions which are stable according to the Ledoux criterion but unstable according to the Schwarzschild criterion. In this series of papers, we analyze the results of an extensive suite of three-dimensional (3D) numerical simulations of the process, and ultimately propose a new 1D prescription for heat and compositional transport in this regime which can be used in stellar or planetary structure and evolution models. In a preliminary study of the phenomenon, Rosenblum et al. showed that, after saturation of the primary instability, a system can evolve in one of two possible ways: the induced turbulence either remains homogeneous, with very weak transport properties, or transitions into a thermo-compositional staircase where the transport rate is much larger (albeit still smaller than in standard convection). In this paper, we show that this dichotomous behavior is a robust property of semi-convection across a wide region of parameter space. We propose a simple semi-analytical criterion to determine whether layer formation is expected or not, and at what rate it proceeds, as a function of the background stratification and of the diffusion parameters (viscosity, thermal diffusivity, and compositional diffusivity) only. The theoretical criterion matches the outcome of our numerical simulations very adequately in the computationally accessible 'planetary' parameter regime and can be extrapolated to the stellar parameter regime. Subsequent papers will address more specifically the question of quantifying transport in the layered case and in the non-layered case.

  6. Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders.

    PubMed

    Wurzman, Rachel; Forcelli, Patrick A; Griffey, Christopher J; Kromer, Lawrence F

    2015-02-01

    EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms. PMID:25281279

  7. Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders

    PubMed Central

    Wurzman, Rachel; Forcelli, Patrick A.; Griffey, Christopher J.; Kromer, Lawrence F.

    2014-01-01

    EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms. PMID:25281279

  8. Bending of Protonema Cells in a Plastid Glycolate/Glycerate Transporter Knockout Line of Physcomitrella patens

    PubMed Central

    Nakahara, Jin; Takechi, Katsuaki; Myouga, Fumiyoshi; Moriyama, Yasuko; Sato, Hiroshi; Takio, Susumu; Takano, Hiroyoshi

    2015-01-01

    Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB–GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO2 conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend. PMID:25793376

  9. Bending of protonema cells in a plastid glycolate/glycerate transporter knockout line of Physcomitrella patens.

    PubMed

    Nakahara, Jin; Takechi, Katsuaki; Myouga, Fumiyoshi; Moriyama, Yasuko; Sato, Hiroshi; Takio, Susumu; Takano, Hiroyoshi

    2015-01-01

    Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB-GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO2 conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend. PMID:25793376

  10. Methylphenidate improves the behavioral and cognitive deficits of neurogranin knockout mice

    PubMed Central

    Huang, Freesia L.; Huang, Kuo-Ping

    2012-01-01

    Neurogranin (Ng), a brain-specific calmodulin-binding protein, is expressed highly in hippocampus, and is important for cognitive function. Deletion of the Ng gene from mice caused attenuation of signal reaction cascade in hippocampus, impairments in learning and memory and high frequency stimulation-induced long-term potentiation. Environmental enrichment alone failed to improve cognitive function. In the present study, behavioral testing revealed that Ng knockout mice were both hyperactive and socially withdrawn. Methylphenidate (MPH) was given to mice while they were also kept under an enrichment condition. MPH treatment reduced the hyperactivity of Ng knockout mice tested in both the open field and forced swim chamber. MPH improved their social abilities such that mice recognized and interacted better with novel subjects. The cognitive memories of MPH-treated mutants were improved in both water maze and contextual fear conditioning tests. High frequency stimulation-induced long-term potentiation of Ng knockout mice was also improved by MPH. The present treatment regimen, however, did not fully reverse the deficits of the mutant mice. In contrast, MPH exerted only a minimal effect on the wild type mice. At the cellular level, MPH increased the number of glial fibrillary acidic protein-positive cells in hippocampus, particularly within the dentate gyrus of Ng knockout mice. Therefore it will be of interest to determine the nature of MPH-mediated astrocyte activation and how it may modulate behavior in future studies. Taken together these Ng knockout mice may be useful for the development of better drug treatment to improve cognitive and behavioral impairments. PMID:22809330

  11. Universal statistics of the knockout tournament

    PubMed Central

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-01-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness. PMID:24217406

  12. Successful second transplantation with non-myeloablative conditioning using haploidentical donors for young patients after graft failure following double umbilical cord cell transplantation.

    PubMed

    Liu, Huilan; Wang, Xingbing; Geng, Liangquan; Tang, Baolin; Tong, Juan; Yao, Wen; Wang, Zuyi; Sun, Zimin

    2010-06-01

    GF is a common and life-threatening complication of UCBT. Here, we report that successful second transplantation of five patients using G-CSF-mobilized maternal stem cells with non-myeloablative conditioning after GF following double UCBT. The median interval between the two transplants were 38 days. The first transplantation was administered after myeloablative conditioning for hematologic malignancies (n=3), and rabbit ATG in combination with cyclophosphamide for SAA (n=2). The second conditioning consisted of Flu and ATG-based non-myeloablative regimen. All five patients acquired quick and sustained engraftment after the second transplant. Treatment-related toxicity was minimal. Three patients developed acute GVHD (>grade II=1). Three patients developed chronic GVHD (limited=1, extensive=2). Severe infectious episodes were significant but manageable. With a median follow-up of 713 days (592-1127), all patients have currently had an event-free survival. These results indicate that a second transplant with non-myeloablative conditioning using mother as the donor for young patient after GF is feasible. PMID:19732379

  13. Finite-amplitude double-component convection due to different boundary conditions for two compensating horizontal gradients

    PubMed

    Tsitverblit

    2000-12-01

    Finite-amplitude convective steady flows that do not bifurcate from the respective conduction state are discovered. They arise as the compensating horizontal gradients of two density-affecting components with equal diffusivities but different boundary conditions are applied to the Boussinesq fluid at rest with and without stable vertical stratification. These flows emanate from convection in a laterally heated stably stratified slot. Their relevance to convective states in a horizontal slot with two vertical gradients, emphasizing universality of the underlying type of convection, is discussed. PMID:11138108

  14. Double Diffusive Magnetohydrodynamic (MHD) Mixed Convective Slip Flow along a Radiating Moving Vertical Flat Plate with Convective Boundary Condition

    PubMed Central

    Rashidi, Mohammad M.; Kavyani, Neda; Abelman, Shirley; Uddin, Mohammed J.; Freidoonimehr, Navid

    2014-01-01

    In this study combined heat and mass transfer by mixed convective flow along a moving vertical flat plate with hydrodynamic slip and thermal convective boundary condition is investigated. Using similarity variables, the governing nonlinear partial differential equations are converted into a system of coupled nonlinear ordinary differential equations. The transformed equations are then solved using a semi-numerical/analytical method called the differential transform method and results are compared with numerical results. Close agreement is found between the present method and the numerical method. Effects of the controlling parameters, including convective heat transfer, magnetic field, buoyancy ratio, hydrodynamic slip, mixed convective, Prandtl number and Schmidt number are investigated on the dimensionless velocity, temperature and concentration profiles. In addition effects of different parameters on the skin friction factor, , local Nusselt number, , and local Sherwood number are shown and explained through tables. PMID:25343360

  15. Human Knockout Carriers: Dead, Diseased, Healthy, or Improved?

    PubMed Central

    Narasimhan, Vagheesh M.; Xue, Yali; Tyler-Smith, Chris

    2016-01-01

    Whole-genome and whole-exome sequence data from large numbers of individuals reveal that we all carry many variants predicted to inactivate genes (knockouts). This discovery raises questions about the phenotypic consequences of these knockouts and potentially allows us to study human gene function through the investigation of homozygous loss-of-function carriers. Here, we discuss strategies, recent results, and future prospects for large-scale human knockout studies. We examine their relevance to studying gene function, population genetics, and importantly, the implications for accurate clinical interpretations. PMID:26988438

  16. Theoretical knock-outs on biological networks.

    PubMed

    Miranda, Pedro J; de S Pinto, Sandro E; Baptista, Murilo S; La Guardia, Giuliano G

    2016-08-21

    In this work we redefine the concept of biological importance and how to compute it, based on a model of complex networks and random walk. We call this new procedure, theoretical knock-out (KO). The proposed method generalizes the procedure presented in a recent study about Oral Tolerance. To devise this method, we make two approaches: algebraically and algorithmically. In both cases we compute a vector on an asymptotic state, called flux vector. The flux is given by a random walk on a directed graph that represents a biological phenomenon. This vector gives us the information about the relative flux of walkers on a vertex which represents a biological agent. With two vector of this kind, we can calculate the relative mean error between them by averaging over its coefficients. This quantity allows us to assess the degree of importance of each vertex of a complex network that evolves in time and has experimental background. We find out that this procedure can be applied in any sort of biological phenomena in which we can know the role and interrelationships of its agents. These results also provide experimental biologists to predict the order of importance of biological agents on a mounted complex network. PMID:27188251

  17. Effect of fuel zoning and fuel nozzle design on pollution emissions at ground idle conditions for a double-annular ram-induction combustor

    NASA Technical Reports Server (NTRS)

    Clements, T. R.

    1973-01-01

    An exhaust emission survey was conducted on a double-annular ram induction combustor at simulated ground idle conditions. The combustor was designed for a large augmented turbofan engine capable of sustained flight speeds up to Mach 3.0. The emission levels of total hydrocarbon (THC), carbon monoxide, carbon dioxide, and nitric oxide were measured. The effects of fuel zoning, fuel nozzle design, and operating conditions (inlet temperature and reference Mach number) on the level of these emissions were determined. At an overall combustor fuel/air ratio of 0.007, fuel zoning reduced THC emissions by a factor of 5 to 1. The reduction in THC emissions is attributed to the increase in local fuel/air ratio provided by the fuel zoning. An alternative method of increasing fuel/air ratio would be to operate with larger-than-normal compressor overboard bleed; however, analysis on this method indicated an increase in idle fuel consumption of 20 percent. The use of air-atomizing nozzles reduced the THC emissions by 2 to 1.

  18. Knockout of the Host Resistance Gene Pkr Fully Restores Replication of Murine Cytomegalovirus m142 and m143 Mutants In Vivo

    PubMed Central

    Ostermann, Eleonore; Warnecke, Gabriele; Waibler, Zoe

    2015-01-01

    Murine cytomegalovirus (MCMV) proteins m142 and m143 are essential for viral replication. They bind double-stranded RNA and prevent protein kinase R-induced protein synthesis shutoff. Whether the two viral proteins have additional functions such as their homologs in human cytomegalovirus do remained unknown. We show that MCMV m142 and m143 knockout mutants attain organ titers equivalent to those attained by wild-type MCMV in Pkr knockout mice, suggesting that these viral proteins do not encode additional PKR-independent functions relevant for pathogenesis in vivo. PMID:26512090

  19. SU-C-BRE-07: Sensitivity Analysis of the Threshold Energy for the Creation of Strand Breaks and of Single and Double Strand Break Clustering Conditions

    SciTech Connect

    Pater, P

    2014-06-15

    Purpose: To analyse the sensitivity of the creation of strand breaks (SB) to the threshold energy (Eth) and thresholding method and to quantify the impact of clustering conditions on single strand break (SSB) and double strand break (DSB) yields. Methods: Monte Carlo simulations using Geant4-DNA were conducted for electron tracks of 280 eV to 220 keV in a geometrical DNA model composed of nucleosomes of 396 phospho-diester groups (PDGs) each. A strand break was created inside a PDG when the sum of all energy deposits (method 1) or energy transfers (method 2) was higher than Eth or when at least one interaction deposited (method 3) or transferred (method 4) an energy higher than Eth. SBs were then clustered into SSBs and DSBs using clustering scoring criteria from the literature and compared to our own. Results: The total number of SBs decreases as Eth is increased. In addition, thresholding on the energy transfers (methods 2 and 4) produces a higher SB count than when thresholding on energy deposits (methods 1 and 3). Method 2 produces a step-like function and should be avoided when attempting to optimize Eth. When SBs are grouped into damage patterns, clustering conditions can underestimated SSBs by up to 18 % and DSBs can be overestimated by up to 12 % compared to our own implementation. Conclusion: We show that two often underreported simulation parameters have a non-negligible effect on overall DNA damage yields. First more SBs are counted when using energy transfers to the PDG rather than energy deposits. Also, SBs grouped according to different clustering conditions can influence reported SSB and DSB by as much as 20%. Careful handling of these parameters is required when trying to compare DNA damage yields from different authors. Research funding from the governments of Canada and Quebec. PP acknowledges partial support by the CREATE Medical Physics Research Training Network grant of the Natural Sciences and Engineering Research Council (Grant number: 432290)

  20. Have gene knockouts caused evolutionary reversals in the mammalian first arch?

    PubMed

    Smith, K K; Schneider, R A

    1998-03-01

    Many recent gene knockout experiments cause anatomical changes to the jaw region of mice that several investigators claim are evolutionary reversals. Here we evaluate these mutant phenotypes and the assertions of atavism. We argue that following the knockout of Hoxa-2, Dlx-2, MHox, Otx2, and RAR genes, ectopic cartilages arise as secondary consequences of disruptions in normal processes of cell specification, migration, or differentiation. These disruptions cause an excess of mesenchyme to accumulate in a region through which skeletal progenitor cells usually migrate, and at a site of condensation that is normally present in mammals but that is too small to chondrify. We find little evidence that these genes, when disrupted, cause a reversion to any primitive condition and although changes in their expression may have played a role in the evolution of the mammalian jaw, their function during morphogenesis is not sufficiently understood to confirm such hypotheses. PMID:9631652

  1. Audiograms, gap detection thresholds, and frequency difference limens in cannabinoid receptor 1 knockout mice.

    PubMed

    Toal, Katrina L; Radziwon, Kelly E; Holfoth, David P; Xu-Friedman, Matthew A; Dent, Micheal L

    2016-02-01

    The cannabinoid receptor 1 (CB1R) is found at several stages in the auditory pathway, but its role in hearing is unknown. Hearing abilities were measured in CB1R knockout mice and compared to those of wild-type mice. Operant conditioning and the psychophysical Method of Constant Stimuli were used to measure audiograms, gap detection thresholds, and frequency difference limens in trained mice using the same methods and stimuli as in previous experiments. CB1R knockout mice showed deficits at frequencies above 8 kHz in their audiograms relative to wild-type mice. CB1R knockouts showed enhancements for detecting gaps in low-pass noisebursts relative to wild-type mice, but were similar for other noise conditions. Finally, the two groups of mice did not differ in their frequency discrimination abilities as measured by the frequency difference limens task. These experiments suggest that the CB1R is involved in auditory processing and lay the groundwork for future physiological experiments. PMID:26427583

  2. Establishment and phenotypic analysis of an Mstn knockout rat.

    PubMed

    Gu, Hao; Cao, Yong; Qiu, Bin; Zhou, Zhiqiang; Deng, Ran; Chen, Zhuang; Li, Rongfeng; Li, Xueling; Wei, Qiang; Xia, Xianzhu; Yong, Weidong

    2016-08-12

    Myostatin (Mstn) is an inhibitor of myogenesis, regulating the number and size of skeletal myocytes. In addition to its myogenic regulatory function, Mstn plays important roles in the development of adipose tissues and in metabolism. In the present study, an Mstn knockout rat model was generated using the zinc finger nuclease (ZFN) technique in order to further investigate the function and mechanism of Mstn in metabolism. The knockout possesses a frame shift mutation resulting in an early termination codon and a truncated peptide of 109 amino acids rather than the full 376 amino acids. The absence of detectable mRNA confirmed successful knockout of Mstn. Relative to wild-type (WT) littermates, Knockout (KO) rats exhibited significantly greater body weight, body circumference, and muscle mass. However, no significant differences in grip force was observed, indicating that Mstn deletion results in greater muscle mass but not greater muscle fiber strength. Additionally, KO rats were found to possess less body fat relative to WT littermates, which is consistent with previous studies in mice and cattle. The aforementioned results indicate that Mstn knockout increases muscle mass while decreasing fat content, leading to observed increases in body weight and body circumference. The Mstn knockout rat model provides a novel means to study the role of Mstn in metabolism and Mstn-related muscle hypertrophy. PMID:27289021

  3. Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice*

    PubMed Central

    Garyali, Punitee; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

    2014-01-01

    Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a−/−, Epm2b−/−, and Epm2a−/− Epm2b−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b−/− and Epm2a−/− Epm2b−/− cells) but not laforin (Epm2a−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. PMID:24914213

  4. Environmental enrichment induces behavioural disturbances in neuropeptide Y knockout mice

    PubMed Central

    Reichmann, Florian; Wegerer, Vanessa; Jain, Piyush; Mayerhofer, Raphaela; Hassan, Ahmed M.; Fröhlich, Esther E.; Bock, Elisabeth; Pritz, Elisabeth; Herzog, Herbert; Holzer, Peter; Leitinger, Gerd

    2016-01-01

    Environmental enrichment (EE) refers to the provision of a complex and stimulating housing condition which improves well-being, behaviour and brain function of laboratory animals. The mechanisms behind these beneficial effects of EE are only partially understood. In the current report, we describe a link between EE and neuropeptide Y (NPY), based on findings from NPY knockout (KO) mice exposed to EE. Relative to EE-housed wildtype (WT) animals, NPY KO mice displayed altered behaviour as well as molecular and morphological changes in amygdala and hippocampus. Exposure of WT mice to EE reduced anxiety and decreased central glucocorticoid receptor expression, effects which were absent in NPY KO mice. In addition, NPY deletion altered the preference of EE items, and EE-housed NPY KO mice responded to stress with exaggerated hyperthermia, displayed impaired spatial memory, had higher hippocampal brain-derived neurotrophic factor mRNA levels and altered hippocampal synaptic plasticity, effects which were not seen in WT mice. Accordingly, these findings suggest that NPY contributes to the anxiolytic effect of EE and that NPY deletion reverses the beneficial effects of EE into a negative experience. The NPY system could thus be a target for “enviromimetics”, therapeutics which reproduce the beneficial effects of enhanced environmental stimulation. PMID:27305846

  5. Modeling fragile X syndrome in the Fmr1 knockout mouse

    PubMed Central

    Kazdoba, Tatiana M.; Leach, Prescott T.; Silverman, Jill L.; Crawley, Jacqueline N.

    2014-01-01

    Summary Fragile X Syndrome (FXS) is a commonly inherited form of intellectual disability and one of the leading genetic causes for autism spectrum disorder. Clinical symptoms of FXS can include impaired cognition, anxiety, hyperactivity, social phobia, and repetitive behaviors. FXS is caused by a CGG repeat mutation which expands a region on the X chromosome containing the FMR1 gene. In FXS, a full mutation (> 200 repeats) leads to hypermethylation of FMR1, an epigenetic mechanism that effectively silences FMR1 gene expression and reduces levels of the FMR1 gene product, fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is important for the regulation of protein expression. In an effort to further understand how loss of FMR1 and FMRP contribute to FXS symptomology, several FXS animal models have been created. The most well characterized rodent model is the Fmr1 knockout (KO) mouse, which lacks FMRP protein due to a disruption in its Fmr1 gene. Here, we review the behavioral phenotyping of the Fmr1 KO mouse to date, and discuss the clinical relevance of this mouse model to the human FXS condition. While much remains to be learned about FXS, the Fmr1 KO mouse is a valuable tool for understanding the repercussions of functional loss of FMRP and assessing the efficacy of pharmacological compounds in ameliorating the molecular and behavioral phenotypes relevant to FXS. PMID:25606362

  6. Environmental enrichment induces behavioural disturbances in neuropeptide Y knockout mice.

    PubMed

    Reichmann, Florian; Wegerer, Vanessa; Jain, Piyush; Mayerhofer, Raphaela; Hassan, Ahmed M; Fröhlich, Esther E; Bock, Elisabeth; Pritz, Elisabeth; Herzog, Herbert; Holzer, Peter; Leitinger, Gerd

    2016-01-01

    Environmental enrichment (EE) refers to the provision of a complex and stimulating housing condition which improves well-being, behaviour and brain function of laboratory animals. The mechanisms behind these beneficial effects of EE are only partially understood. In the current report, we describe a link between EE and neuropeptide Y (NPY), based on findings from NPY knockout (KO) mice exposed to EE. Relative to EE-housed wildtype (WT) animals, NPY KO mice displayed altered behaviour as well as molecular and morphological changes in amygdala and hippocampus. Exposure of WT mice to EE reduced anxiety and decreased central glucocorticoid receptor expression, effects which were absent in NPY KO mice. In addition, NPY deletion altered the preference of EE items, and EE-housed NPY KO mice responded to stress with exaggerated hyperthermia, displayed impaired spatial memory, had higher hippocampal brain-derived neurotrophic factor mRNA levels and altered hippocampal synaptic plasticity, effects which were not seen in WT mice. Accordingly, these findings suggest that NPY contributes to the anxiolytic effect of EE and that NPY deletion reverses the beneficial effects of EE into a negative experience. The NPY system could thus be a target for "enviromimetics", therapeutics which reproduce the beneficial effects of enhanced environmental stimulation. PMID:27305846

  7. Conditions required for the appearance of double responses in hippocampal field CA1 to application of single stimuli to Shäffer collaterals in freely moving rats.

    PubMed

    Zosimovskii, V A; Korshunov, V A; Markevich, V A

    2008-03-01

    Stimulation of Shäffer collaterals with single current impulses could evoke double responses in hippocampal field CA1 in freely moving rats. The late response - the population excitatory postsynaptic potential with a preceding transient potential, often biphasic - occurred only after an early population spike and was time-locked to it. The shape characteristics of the late response, its polarity, and its latent period relative to the early population spike suggest that stimulation of Shäffer collaterals gives rise, in CA1, to a wave of excitation which passes through the entorhinal cortex and returns to CA1 directly via fibers of the perforant path. In conscious rats, medium-strength stimulation of Shäffer collaterals, sufficient to evoke a quite early population spike in CA1, did not usually lead to the appearance of a late response; the same stimulation became effective after tetanization of Shäffer collaterals in conditions of long-term potentiation of the early population spike. Furthermore, the appearance of the late response was facilitated in rats falling asleep on the background of high-amplitude, low-frequency EEG oscillations in CA1 characteristic of slow-wave sleep, as well as in sleeping rats, regardless of the EEG pattern. PMID:18264779

  8. [Conditions required for appearance of a double response to a single-shock stimulation of Schaffer collaterals in hippocampal field CA1 in freely moving rats].

    PubMed

    Zosimovskiĭ, V A; Korshunov, V A; Markevich, V A

    2007-01-01

    Schaffer collateral stimulation with a single current impulse can evoke a double response in hippocampal field CA1 of freely moving rats. The late response appears as a population excitatory postsynaptic potential with a preceding short-term potential (frequently biphasic) only after the early population spike and is time-locked to it. The wave shape and polarity of the late response, its latency with respect to the peak of the early population spike suggest that the excitation wave produced in the CA1 field by the stimulation of Schaffer collaterals passes across the entorhinal cortex and returns to the CA1 directly via the perforant path fibers. In waking rat, the medium-intensity stimulation of Schaffer collaterals (able to evoke in the CA1 an early population spike of sufficiently high amplitude) usually does not result in the appearance of the late response. However, similar stimulation becomes efficient after the tetanization of Schaffer collaterals, under conditions of the long-term potentiation of the early population spike. Moreover, the late response occurrence is facilitated in a rat falling asleep after the development in the CA1 of high-amplitude low-frequency EEG oscillations typical for the slow-wave sleep and in a sleeping rat independently of the EEG pattern. PMID:17596017

  9. Skin-whitening and skin-condition-improving effects of topical oxidized glutathione: a double-blind and placebo-controlled clinical trial in healthy women

    PubMed Central

    Watanabe, Fumiko; Hashizume, Erika; Chan, Gertrude P; Kamimura, Ayako

    2014-01-01

    Purpose Glutathione is a tripeptide consisting of cysteine, glycine, and glutamate and functions as a major antioxidant. It is synthesized endogenously in humans. Glutathione protects thiol protein groups from oxidation and is involved in cellular detoxification for maintenance of the cell environment. Reduced glutathione (GSH) has a skin-whitening effect in humans through its tyrosinase inhibitory activity, but in the case of oxidized glutathione (GSSG) this effect is unclear. We examined the skin-whitening and skin-condition effects of topical GSSG in healthy women. Subjects and methods The subjects were 30 healthy adult women aged 30 to 50 years. The study design was a randomized, double-blind, matched-pair, placebo-controlled clinical trial. Subjects applied GSSG 2% (weight/weight [w/w]) lotion to one side of the face and a placebo lotion to the other side twice daily for 10 weeks. We objectively measured changes in melanin index values, moisture content of the stratum corneum, smoothness, wrinkle formation, and elasticity of the skin. The principal investigator and each subject also used subjective scores to investigate skin whitening, wrinkle reduction, and smoothness. Analysis of variance was used to evaluate differences between groups. Results The skin melanin index was significantly lower with GSSG treatment than with placebo from the early weeks after the start of the trial through to the end of the study period (at 10 weeks, P<0.001). In addition, in the latter half of the study period GSSG-treated sites had significant increases in moisture content of the stratum corneum, suppression of wrinkle formation, and improvement in skin smoothness. There were no marked adverse effects from GSSG application. Conclusion Topical GSSG is safe and effectively whitens the skin and improves skin condition in healthy women. PMID:25378941

  10. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences

    SciTech Connect

    Cai, Yuping; Wolk, C.P. )

    1990-06-01

    Use of the sacB gene provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, they mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

  11. Organic anion transporter 3 (Oat3/Slc22a8) knockout mice exhibit altered clearance and distribution of penicillin G

    PubMed Central

    VanWert, Adam L.; Bailey, Rachel M.; Sweet, Douglas H.

    2010-01-01

    The interaction of renal basolateral organic anion transporter 3 (Oat3) with commonly used pharmacotherapeutics (e.g., NSAIDs, β-lactams, and methotrexate) has been studied extensively in vitro. However, the in vivo role of Oat3 in drug disposition, in the context of other transporters, glomerular filtration, and metabolism, has not been established. Moreover, recent investigations have identified inactive human OAT3 polymorphisms. Therefore, this investigation was designed to elucidate the in vivo role of Oat3 in the disposition of penicillin G and prototypical substrates using an Oat3 knockout mouse model. Oat3 deletion resulted in a doubling of penicillin’s half-life (P < 0.05) and a reduced volume of distribution (P < 0.01), together yielding a plasma clearance that was one-half (P < 0.05, males) to one-third (P < 0.001, females) of that in wild-type mice. Inhibition of Oat3 abolished the differences in penicillin G elimination between genotypes. Hepatic accumulation of penicillin was 2.3 times higher in male knockouts (P < 0.05) and 3.7 times higher in female knockouts (P < 0.001). Female knockouts also exhibited impaired estrone-3-sulfate clearance. Oat3 deletion did not impact p-aminohippurate elimination, providing correlative evidence to studies in Oat1 knockout mice that suggest Oat1 governs tubular uptake of p-aminohippurate. Collectively, these findings are the first to indicate that functional Oat3 is necessary for proper elimination of xenobiotic and endogenous compounds in vivo. Thus Oat3 plays a distinct role in determining the efficacy and toxicity of drugs. Dysfunctional human OAT3 polymorphisms or instances of polypharmacy involving OAT3 substrates may result in altered systemic accumulation of β-lactams and other clinically relevant compounds. PMID:17686950

  12. GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

    PubMed

    Watanabe, Takahito; Noji, Sumihare; Mito, Taro

    2016-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. PMID:26443220

  13. Single, double, and triple Auger decay probabilities of C+(1 s 2 s22 p22 D ,2 P ) resonances

    NASA Astrophysics Data System (ADS)

    Zhou, Fuyang; Ma, Yulong; Qu, Yizhi

    2016-06-01

    Single, double, and triple Auger decay rates of C+(1 s 2 s22 p22 D ,2 P ) resonances were calculated in the framework of perturbation theory. The direct double Auger decay probabilities were calculated by using the approximate formulas according to the knockout and shakeoff mechanisms, in which the knockout mechanism was found to be dominant. Then the knockout mechanism was employed to investigate the complex triple Auger decay process, and the calculated rates have good agreement with the available experimental values.

  14. Knockout Mice Challenge our Concepts of Glucose Homeostasis and the Pathogenesis of Diabetes

    PubMed Central

    2003-01-01

    A central component of type 2 diabetes and the metabolic syndrome is insulin resistance. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems. Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway provides a unique approach to determining the role of individual proteins in the molecular mechanism of insulin action and the pathogenesis of insulin resistance and diabetes. The role of the four major insulin receptor substrates (IRS1-4) in insulin and IGF-1 signaling have been examined by creating mice with targeted gene knockouts. Each produces a unique phenotype, indicating the complementary role of these signaling components. Combined heterozygous defects often produce synergistic or epistatic effects, although the final severity of the phenotype depends on the genetic background of the mice. Conditional knockouts of the insulin receptor have also been created using the Cre-lox system. These tissue specific knockouts have provide unique insights into the control of glucose homeostasis and the pathogenesis of type 2 diabetes, and have led to development of new hypotheses about the nature of the insulin action and development of diabetes. PMID:15061645

  15. The alcohol-induced locomotor stimulation and accumbal dopamine release is suppressed in ghrelin knockout mice.

    PubMed

    Jerlhag, Elisabet; Landgren, Sara; Egecioglu, Emil; Dickson, Suzanne L; Engel, Jörgen A

    2011-06-01

    Ghrelin, the first endogenous ligand for the type 1A growth hormone secretagogue receptor (GHS-R1A), plays a role in energy balance, feeding behavior, and reward. Previously, we showed that pharmacologic and genetic suppression of the GHS-R1A attenuates the alcohol-induced stimulation, accumbal dopamine release, and conditioned place preference as well as alcohol consumption in mice, implying that the GHS-R1A is required for alcohol reward. The present study further elucidates the role of ghrelin for alcohol-induced dopamine release in nucleus accumbens and locomotor stimulation by means of ghrelin knockout mice. We found that the ability of alcohol to increase accumbal dopamine release in wild-type mice is not observed in ghrelin knockout mice. Furthermore, alcohol induced a locomotor stimulation in the wild-type mice and ghrelin knockout mice; however, the locomotor stimulation in homozygote mice was significantly lower than in the wild-type mice. The present series of experiments suggest that endogenous ghrelin may be required for the ability of alcohol to activate the mesolimbic dopamine system. PMID:21145690

  16. Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effector nuclease- (TALEN)-mediated gene inactivation

    PubMed Central

    Ferguson, Carolyn; McKay, Matthew; Harris, R. Adron; Homanics, Gregg E.

    2013-01-01

    Genetically engineered mice are a valuable resource for studies of the behavioral effects of ethanol. However, for some behavioral tests of ethanol action, the rat is a superior model organism. Production of genetically engineered rats has been severely hampered due to technical limitations. Here we utilized a promising new technique for efficient site-specific gene modification to create a novel gene knockout rat line. This approach is based on Transcriptional Activator-Like Effector Nucleases (TALENs). TALENs function in pairs and bind DNA in a sequence-specific manner. Upon binding to the target sequence, a functional nuclease is reconstituted that creates double-stranded breaks in the DNA that are efficiently repaired by non-homologous end joining. This error-prone process often results in deletions of varying lengths at the targeted locus. The toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and neurodegeneration, as well as multiple ethanol-induced behavioral effects. To generate Tlr4 knockout rats, a pair of TALEN constructs was created that specifically target Exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. The mutated allele consisted of a 13 base-pair deletion that was predicted to create a frameshift mutation after amino acid 25. This founder rat successfully transmitted the mutation to F1 offspring. Heterozygous F1 offspring were interbred to produce homozygous F2 animals. Homozygous mutants expressed the 13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. Thus, the mutant Tlr4 allele generated by TALEN-mediated gene

  17. Cathepsin K knockout alleviates aging-induced cardiac dysfunction

    PubMed Central

    Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

    2015-01-01

    Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

  18. Vibroacoustical behaviour of multilayered heterogeneous plates with elastic support and interface condition: Application to the case of the double-deck French high- speed train

    NASA Astrophysics Data System (ADS)

    Chabaud, Thierry Rene

    1998-10-01

    Mass Transit has to meet specifications of increasing difficulty which put in the forefront areas which were before considered as secondary. In particular, passengers acoustic comfort has become a major criterion, reflecting the requirements for quality transportation. The present work has its origin in this specific context and more specially in the intention of FAIVELEY TRANSPORT, a Railway equipment supplier, to improve the acoustic behaviour of its products. The aim of this study is the comprehension of the physical phenomena of the noise transmission through the doors of the transport vehicles, from outside to inside. The Railway access doors are the main application of this work. The considered frequency range is low frequencies below 500 Hz. The final objective is to notably reduce the transmitted noise in order to improve the passengers' acoustic comfort inside the transport vehicles. To do this, we developed a model based on an analytical integro-modal approach, associated with a Rayleigh-Ritz approximation. The original aspect of this work is to develop a model of a vibrating structure which take into account its multilayered and heterogeneous aspects and its complex boundary and interface conditions (linear and punctual elastic stiffness). This model is the basis of a specific software for preliminary design studies (VANTAIL) which is able to indicate, with a parametric study, the contribution of each different part of the doors to the global vibroacoustic behaviour. An experimental study on an industrial structure (the access door of the double deck French high speed train) permits us to validate the developed software, to define its limits and to validate the proposed vibroacoustic treatments.

  19. Effects of extreme thermal conditions on plasticity in breeding phenology and double-broodedness of Great Tits and Blue Tits in central Poland in 2013 and 2014

    NASA Astrophysics Data System (ADS)

    Glądalski, Michał; Bańbura, Mirosława; Kaliński, Adam; Markowski, Marcin; Skwarska, Joanna; Wawrzyniak, Jarosław; Zieliński, Piotr; Bańbura, Jerzy

    2016-03-01

    Many avian species in Europe breed earlier as a result of higher temperatures caused by global climate changes. Climate change means not only higher temperatures but also more frequent extreme weather events, sometimes contrasting with the long-term trends. It was suggested that we should look closely at every extreme phenomenon and its consequences for the phenology of organisms. Examining the limits of phenotypic plasticity may be an important goal for future research. Extremely low spring temperatures in 2013 (coldest spring in 40 years) resulted in birds laying unusually late, and it was followed in 2014 by the earliest breeding season on record (warmest spring in 40 years). Here, we present results concerning breeding phenology and double-broodedness in the Great Tit (Parus major) and the Blue Tit (Cyanistes caeruleus) in 2013 and 2014 in an urban parkland and a deciduous forest in central Poland. Great Tits started laying eggs 18.2 days later in 2013 than in 2014 in the parkland, whereas the analogous difference was 21.1 days in the forest. Blue Tits started laying eggs in the parkland 18.5 days later in 2013 than in 2014, while the analogous difference was 21.6 days in the forest. The difference in the proportion of second clutches in Great Tits between 2013 (fewer second clutches) and 2014 (more second clutches) was highly significant in the parkland and in the forest. This rather large extent of breeding plasticity has developed in reaction to challenges of irregular inter-annual variability of climatic conditions. Such a buffer of plasticity may be sufficient for Blue Tits and Great Tits to adjust the timing of breeding to the upcoming climate changes.

  20. CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

    PubMed Central

    Shinmyo, Yohei; Tanaka, Satoshi; Tsunoda, Shinichi; Hosomichi, Kazuyoshi; Tajima, Atsushi; Kawasaki, Hiroshi

    2016-01-01

    The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. PMID:26857612

  1. Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice

    PubMed Central

    Hiebert, P R; Wu, D; Granville, D J

    2013-01-01

    Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM. PMID:23912712

  2. Generation and characterization of RAG2 knockout pigs as animal model for severe combined immunodeficiency.

    PubMed

    Suzuki, Shunichi; Iwamoto, Masaki; Hashimoto, Michiko; Suzuki, Misae; Nakai, Michiko; Fuchimoto, Daiichiro; Sembon, Shoichiro; Eguchi-Ogawa, Tomoko; Uenishi, Hirohide; Onishi, Akira

    2016-10-01

    Pigs with severe combined immunodeficiency (SCID) are versatile animal models for human medical research because of their biological similarities to humans, suitable body size, and longevity for practical research. SCID pigs with defined mutation(s) can be an invaluable tool for research on porcine immunity. In this study, we produced RAG2-knockout pigs via somatic cell nuclear transfer and analyzed their phenotype. The V(D)J recombination processes were confirmed as being inactivated. They consistently lacked mature T and B cells but had substantial numbers of cells considered to be T- or B-cell progenitors as well as NK cells. They also lacked thymic medulla and lymphoid aggregations in the spleen, mesenteric lymph nodes, and ileal Peyer's patches. We showed more severe immunological defects in the RAG2 and IL2RG double-knockout pig through this study. Thus, SCID pigs could be promising animal models not only for translational medical research but also for immunological studies of pigs themselves. PMID:27496741

  3. A new ecamsule-containing SPF 40 sunscreen cream for the prevention of polymorphous light eruption: a double-blind, randomized, controlled study in maximized outdoor conditions.

    PubMed

    DeLeo, Vincent A; Clark, Scott; Fowler, Joseph; Poncet, Michel; Loesche, Christian; Soto, Pascale

    2009-02-01

    Polymorphous light eruption (PMLE) is an idiopathic photodermatosis elicited by UV radiation (UVR). The objective of this double-blind, randomized, controlled, intraindividual, bilateral comparison was to determine the efficacy of the UVA filters (ecamsule, avobenzone) present in the new sun protection factor (SPF) 40 sunscreen cream in preventing PMLE in maximized outdoor conditions (ie, exaggerated sun exposure). Safety also was assessed. Each participant was treated with SPF 40 sunscreen cream containing ecamsule 3%, octocrylene 10%, avobenzone 2%, and titanium dioxide 5% (tetrad) on one side of the body and either an ecamsule-deprived (triad-E) or avobenzone-deprived (triad-A) cream on the other side. Participants were subsequently exposed to incremental doses of sunlight for up to 6 days. The primary efficacy assessment was a composite relative success rate with 3 components. Success was defined as either a delayed time to onset of PMLE or a lower global severity of PMLE comparing one side of the body to the other side in the same participant. Safety evaluations included systemic adverse events (AEs). Of the 144 participants enrolled and randomized, 22 did not experience PMLE during the study duration under these maximized sun exposure conditions. A significantly greater number of successes were detected on the tetrad-treated side compared with either triad: 41 of 73 participants (56%) versus 8 of 73 participants (11%; P<.001) in the triad-E treatment group and 26 of 71 participants (36%) versus 11 of 71 participants (16%; P=.02) in the triad-A treatment group. Polymorphous light eruption appeared later with the tetrad than with either triad. The global severity of the PMLE flares was significantly lower with the tetrad than with both triads at end point (P<.001 and P=.02 for tetrad vs triad-E and tetrad vs triad-A, respectively). In this study, the SPF 40 sunscreen cream containing ecamsule 3%, octocrylene 10%, avobenzone 2%, and titanium dioxide 5% prevented

  4. Salty taste deficits in CALHM1 knockout mice.

    PubMed

    Tordoff, Michael G; Ellis, Hillary T; Aleman, Tiffany R; Downing, Arnelle; Marambaud, Philippe; Foskett, J Kevin; Dana, Rachel M; McCaughey, Stuart A

    2014-07-01

    Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt. PMID:24846212

  5. Maximal Oxygen Consumption Is Reduced in Aquaporin-1 Knockout Mice.

    PubMed

    Al-Samir, Samer; Goossens, Dominique; Cartron, Jean-Pierre; Nielsen, Søren; Scherbarth, Frank; Steinlechner, Stephan; Gros, Gerolf; Endeward, Volker

    2016-01-01

    We have measured maximal oxygen consumption ([Formula: see text]O2,max) of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9, and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that [Formula: see text]O2,max as determined by the Helox technique is reduced by ~16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of [Formula: see text]O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2) by pulse oximetry. Neither under normoxic (inspiratory O2 21%) nor under hypoxic conditions (11% O2) is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of [Formula: see text]O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of [Formula: see text]O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced [Formula: see text]O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced [Formula: see text]O2,max, which constitutes a new phenotype of these mice. PMID:27559317

  6. Maximal Oxygen Consumption Is Reduced in Aquaporin-1 Knockout Mice

    PubMed Central

    Al-Samir, Samer; Goossens, Dominique; Cartron, Jean-Pierre; Nielsen, Søren; Scherbarth, Frank; Steinlechner, Stephan; Gros, Gerolf; Endeward, Volker

    2016-01-01

    We have measured maximal oxygen consumption (V˙O2,max) of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9, and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that V˙O2,max as determined by the Helox technique is reduced by ~16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of V˙O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2) by pulse oximetry. Neither under normoxic (inspiratory O2 21%) nor under hypoxic conditions (11% O2) is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of V˙O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of V˙O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced V˙O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced V˙O2,max, which constitutes a new phenotype of these mice. PMID:27559317

  7. P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis

    PubMed Central

    Viering, Daan H. H. M.; Bos, Caro; Bindels, René J. M.; Hoenderop, Joost G. J.

    2016-01-01

    ATP-mediated signaling is an important regulator of electrolyte transport in the kidney. The purinergic cation channel P2X6 has been previously localized to the distal convoluted tubule (DCT), a nephron segment important for Mg2+ and Na+ reabsorption, but its role in ion transport remains unknown. In this study, P2x6 knockout (P2x6-/-) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The P2x6-/- animals displayed a normal phenotype and did not differ physiologically from wild type mice. Differences in serum concentration and 24-hrs urine excretion of Na+, K+, Mg2+ and Ca2+ were not detected between P2x6+/+, P2x6+/- and P2x6-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other P2x subunits and electrolyte transporters, including P2x1-5, P2x7, Trpm6, Ncc, Egf, Cldn16, Scnn1, Slc12a3, Slc41a1, Slc41a3, Cnnm2, Kcnj10 and Fxyd2. Additionally, protein levels of P2X2 and P2X4 were assessed in P2x6+/+ and P2x6-/- mouse kidneys. However, significant changes in expression were not detected. Furthermore, no compensatory changes in gene expression could be demonstrated in heart material isolated from P2x6-/- mice. Except for a significant (P<0.05) upregulation of P2x2 in the heart of P2x6-/- mice compared to the P2x6+/+ mice. Thus, our data suggests that purinergic signaling via P2X6 is not significantly involved in the regulation of renal electrolyte handling under normal physiological conditions. PMID:27254077

  8. Comprehensive Behavioral Analysis of Cluster of Differentiation 47 Knockout Mice

    PubMed Central

    Koshimizu, Hisatsugu; Takao, Keizo; Matozaki, Takashi; Ohnishi, Hiroshi; Miyakawa, Tsuyoshi

    2014-01-01

    Cluster of differentiation 47 (CD47) is a member of the immunoglobulin superfamily which functions as a ligand for the extracellular region of signal regulatory protein α (SIRPα), a protein which is abundantly expressed in the brain. Previous studies, including ours, have demonstrated that both CD47 and SIRPα fulfill various functions in the central nervous system (CNS), such as the modulation of synaptic transmission and neuronal cell survival. We previously reported that CD47 is involved in the regulation of depression-like behavior of mice in the forced swim test through its modulation of tyrosine phosphorylation of SIRPα. However, other potential behavioral functions of CD47 remain largely unknown. In this study, in an effort to further investigate functional roles of CD47 in the CNS, CD47 knockout (KO) mice and their wild-type littermates were subjected to a battery of behavioral tests. CD47 KO mice displayed decreased prepulse inhibition, while the startle response did not differ between genotypes. The mutants exhibited slightly but significantly decreased sociability and social novelty preference in Crawley’s three-chamber social approach test, whereas in social interaction tests in which experimental and stimulus mice have direct contact with each other in a freely moving setting in a novel environment or home cage, there were no significant differences between the genotypes. While previous studies suggested that CD47 regulates fear memory in the inhibitory avoidance test in rodents, our CD47 KO mice exhibited normal fear and spatial memory in the fear conditioning and the Barnes maze tests, respectively. These findings suggest that CD47 is potentially involved in the regulation of sensorimotor gating and social behavior in mice. PMID:24586890

  9. Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV)

    PubMed Central

    Laidlaw, Stephen M.; Skinner, Michael A.

    2016-01-01

    The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.

  10. Monocular Elevation Deficiency - Double Elevator Palsy

    MedlinePlus

    ... Eye Terms Conditions Frequently Asked Questions Español Condiciones Chinese Conditions Monocular Elevation Deficiency/ Double Elevator Palsy En Español Read in Chinese What is monocular elevation deficiency (Double Elevator Palsy)? ...

  11. An analysis of T cell intrinsic roles of E2A by conditional gene disruption in the thymus.

    PubMed

    Pan, Lihua; Hanrahan, Jenifer; Li, Jie; Hale, Laura P; Zhuang, Yuan

    2002-04-15

    The importance of E2A transcription factors in T cell development has been demonstrated in studies of E2A-deficient mice, which display abnormal T cell development and a high frequency of T cell lymphomas. Because E2A expression is not restricted to the T cell lineage, the primary cause of the T cell phenotype in E2A-deficient mice was not fully determined. To further investigate the role of E2A in T cell lineage, we generated mice with the E2A gene disrupted exclusively during thymocyte development using the Cre-lox system. We show that this system allows E2A gene disruption to occur throughout the double-negative stage of thymocyte development. E2A deletion appears to be completed before development reaches the double-positive stage. Consistent with the gene disruption, these mice reveal a T cell intrinsic role for E2A during the transition from the double-negative stage to the double-positive stage of thymocyte development. In contrast to germline E2A knockout mice, conditional E2A knockout mice do not develop T cell lymphoma. This work establishes a new model for further investigating E2A function in T cell development and leukemiogenesis. PMID:11937548

  12. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology

    PubMed Central

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  13. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology.

    PubMed

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  14. The Impact of Remote Ischemic Pre-Conditioning on Contrast-Induced Nephropathy in Patients Undergoing Coronary Angiography and Angioplasty: A Double-Blind Randomized Clinical Trial

    PubMed Central

    Gholoobi, Arash; Sajjadi, Seyyed Masoud; Shabestari, Mahmoud Mohammadzadeh; Eshraghi, Ali; Shamloo, Alireza Sepehri

    2015-01-01

    Background and objective Contrast-induced nephropathy (CIN) is an acute major complication following intravascular administration of iodinated contrast agents; however, the best approach for preventing CIN is not clear. Remote ischemic pre-conditioning (RIPC) is a new, non-pharmacological method that has been considered for the prevention of CIN following coronary angiography. This study assessed the effects of RIPC with four brief episodes of upper limb ischemia and reperfusion in the prevention of contrast-induced nephropathy (CIN) after coronary angiography and/or angioplasty. Methods In this double-blind randomized clinical trial, we enrolled 51 patients with chronic stable angina and non-ST elevation acute coronary syndrome (NSTE.ACS), and they underwent coronary angiography and/or angioplasty. Standard fluid therapy with normal saline was prescribed for all patients before and after the procedure. The patients were divided into two groups, i.e., a study group of patients who had undergone RIPC intervention and a control group of patients who had not undergone RIPC. One hour before the procedure, a sphygmomanometer cuff was placed around one arm and inflated up to 50 mmHg above the systolic pressure for five minutes; then, the cuff was deflated for another five minutes, and this cycle was repeated four times. The patients’ serum creatinine levels were measured at baseline and 48 hours after the procedure, and the incidence of CIN was calculated. Results Twenty-one males and 30 females were studied in two groups, i.e., an RIPC intervention group (n = 25) and a control group (n = 26) that were homogenous considering baseline characteristics. No significant difference was observed in the mean level of serum creatinine between the two groups at a post-intervention time of 48 hours (RICP: 1.74 ± 0.70 mg/dL vs. Control: 1.75 ± 0.87 mg/dL; P = 0.64). However, a lower incidence rate of CIN was observed 48 hours after the administration of the contrast medium in

  15. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

    PubMed

    Chu, H W; Rios, C; Huang, C; Wesolowska-Andersen, A; Burchard, E G; O'Connor, B P; Fingerlin, T E; Nichols, D; Reynolds, S D; Seibold, M A

    2015-10-01

    Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. PMID:26043872

  16. [A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].

    PubMed

    Fenghua, Zhang; Houpeng, Wang; Siyu, Huang; Feng, Xiong; Zuoyan, Zhu; Yonghua, Sun

    2016-02-01

    Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions. PMID:26907778

  17. One-neutron knockout from 51-55 Sc

    NASA Astrophysics Data System (ADS)

    Schwertel, S.; Maierbeck, P.; Krücken, R.; Gernhäuser, R.; Kröll, T.; Alvarez-Pol, H.; Aksouh, F.; Aumann, T.; Behr, K.; Benjamim, E. A.; Benlliure, J.; Bildstein, V.; Böhmer, M.; Boretzky, K.; Borge, M. J. G.; Brünle, A.; Bürger, A.; Caamaño, M.; Casarejos, E.; Chatillon, A.; Chulkov, L. V.; Cortina-Gil, D.; Enders, J.; Eppinger, K.; Faestermann, T.; Friese, J.; Fabbietti, L.; Gascón, M.; Geissel, H.; Gerl, J.; Gorska, M.; Hansen, P. G.; Jonson, B.; Kanungo, R.; Kiselev, O.; Kojouharov, I.; Klimkiewicz, A.; Kurtukian, T.; Kurz, N.; Larsson, K.; Le Bleis, T.; Mahata, K.; Maier, L.; Nilsson, T.; Nociforo, C.; Nyman, G.; Pascual-Izarra, C.; Perea, A.; Perez, D.; Prochazka, A.; Rodriguez-Tajes, C.; Rossi, D.; Schaffner, H.; Schrieder, G.; Simon, H.; Sitar, B.; Stanoiu, M.; Sümmerer, K.; Tengblad, O.; Weick, H.; Winkler, S.; Brown, B. A.; Otsuka, T.; Tostevin, J. A.; Rae, W. D. M.

    2012-12-01

    Results are presented from a one-neutron knockout experiment at relativistic energies of ≈ 420 A MeV on 51-55Sc using the GSI Fragment Separator as a two-stage magnetic spectrometer and the MINIBALL array for gamma-ray detection. Inclusive longitudinal momentum distributions and cross-sections were measured enabling the determination of the contributions corresponding to knockout from the ν p_{1/2} , ν p_{3/2} , ( L = 1 and ν f_{7/2} , ν f_{5/2} ( L = 3 neutron orbitals. The observed L = 1 and L = 3 contributions are compared with theoretical cross-sections using eikonal knockout theory and spectroscopic factors from shell model calculations using the GXPF1A interaction. The measured inclusive knockout cross-sections generally follow the trends expected theoretically and given by the spectroscopic strength predicted from the shell model calculations. However, the deduced L = 1 cross-sections are generally 30-40% higher while the L = 3 contributions are about a factor of two smaller than predicted. This points to a promotion of neutrons from the ν f_{7/2} to the ν p_{3/2} orbital indicating a weakening of the N = 28 shell gap in these nuclei. While this is not predicted for the phenomenological GXPF1A interaction such a weakening is predicted by recent calculations using realistic low-momentum interactions V_{low k} obtained by evolving a chiral N3LO nucleon-nucleon potential.

  18. Photocatalytic organic transformation by layered double hydroxides: highly efficient and selective oxidation of primary aromatic amines to their imines under ambient aerobic conditions.

    PubMed

    Yang, Xiu-Jie; Chen, Bin; Li, Xu-Bing; Zheng, Li-Qiang; Wu, Li-Zhu; Tung, Chen-Ho

    2014-06-25

    We report the first application of layered double hydroxide as a photocatalyst in the transformation of primary aromatic amines to their corresponding imines with high efficiency and selectivity by using oxygen in an air atmosphere as a terminal oxidant under light irradiation. PMID:24827163

  19. Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

  20. Deleting both PHLPP1 and CANP1 rescues impairments in long-term potentiation and learning in both single knockout mice.

    PubMed

    Liu, Yan; Sun, Jiandong; Wang, Yubin; Lopez, Dulce; Tran, Jennifer; Bi, Xiaoning; Baudry, Michel

    2016-08-01

    Calpain-1 (CANP1) has been shown to play a critical role in synaptic plasticity and learning and memory, as its deletion in mice results in impairment in theta-burst stimulation- (TBS) induced LTP and various forms of learning and memory. Likewise, PHLPP1 (aka SCOP) has also been found to participate in learning and memory, as PHLPP1 overexpression impairs hippocampus-dependent learning. We previously showed that TBS-induced LTP was associated with calpain-1 mediated truncation of PHLPP1.To better understand the roles of these 2 genes in synaptic plasticity and learning and memory, we generated a double knockout (DKO) mouse by crossing the parent strains. Surprisingly, DKO mice exhibit normal TBS-induced LTP, and the learning impairments in fear conditioning and novel object or novel location recognition were absent in the DKO mice. Moreover, TBS-induced ERK activation in field CA1 of hippocampal slices, which is impaired in both single deletion mice, was restored in the DKO mice. These results further strengthen the roles of both CANP1 and PHLPP1 in synaptic plasticity and learning and memory, and illustrate the complexities of the interactions between multiple pathways participating in synaptic plasticity. PMID:27421891

  1. Behavioral characterization of P311 knockout mice

    PubMed Central

    Taylor, Gregory A.; Rodriguiz, Ramona M.; Greene, Robert I.; Daniell, Xiaoju; Henry, Stanley C.; Crooks, Kristy R.; Kotloski, Robert; Tessarollo, Lino; Phillips, Lindsey E.; Wetsel, William C.

    2013-01-01

    P311 is an 8-kDa protein that is expressed in many brain regions, particularly the hippocampus, cerebellum and olfactory lobes, and is under stringent regulation by developmental, mitogenic and other physiological stimuli. P311 is thought to be involved in the transformation and motility of neural cells; however, its role in normal brain physiology is undefined. To address this point, P311-deficient mice were developed through gene targeting and their behaviors were characterized. Mutants displayed no overt abnormalities, bred normally and had normal survival rates. Additionally, no deficiencies were noted in motor co-ordination, balance, hearing or olfactory discrimination. Nevertheless, P311-deficient mice showed altered behavioral responses in learning and memory. These included impaired responses in social transmission of food preference, Morris water maze and contextual fear conditioning. Additionally, mutants displayed altered emotional responses as indicated by decreased freezing in contextual and cued fear conditioning and reduced fear-potentiated startle. Together, these data establish P311 as playing an important role in learning and memory processes and emotional responses. PMID:18616608

  2. Spectroscopy of 23F by quasi-free proton knockout reaction

    NASA Astrophysics Data System (ADS)

    Tang, Tsz Leung

    2014-09-01

    The separation energy of quasi-free proton knockout reactioncan be a good probe for the single particle energy of each orbit inside a nucleus. The spectroscopic factor can also bededuced from the measured cross section. The effective single particle energy (ESPE)can then be calculated as a spectroscopic factor weighted mean of single particle energy. The ESPE can reveal the strength of spin-orbit splitting. This splitting is further related to the effects of tensor force, 3N force and spin-orbit coupling of nuclear force. Florine has 1 proton on the s-d shell. The single particle picture should be suitable to explain its behavior. However, in the neutron rich isotopes, this picture may be broken due to the effect of excessive neutrons on the s-d shell. The possible effects are s-d shell mixing and reduction of shell gap energy. We are going to present the experimental setup and condition, data analysis process and the latest data analysis result for exclusive measurement of F(p,2p)O* knockout reaction. The excitation energy spectrum of residual nucleus will be discussed. The yield will be compared with the theoretical calculation of the cross section by code THREEDEE. The separation energy of quasi-free proton knockout reactioncan be a good probe for the single particle energy of each orbit inside a nucleus. The spectroscopic factor can also bededuced from the measured cross section. The effective single particle energy (ESPE)can then be calculated as a spectroscopic factor weighted mean of single particle energy. The ESPE can reveal the strength of spin-orbit splitting. This splitting is further related to the effects of tensor force, 3N force and spin-orbit coupling of nuclear force. Florine has 1 proton on the s-d shell. The single particle picture should be suitable to explain its behavior. However, in the neutron rich isotopes, this picture may be broken due to the effect of excessive neutrons on the s-d shell. The possible effects are s-d shell mixing and

  3. Appetitively motivated instrumental learning in SynGAP heterozygous knockout mice.

    PubMed

    Muhia, Mary; Feldon, Joram; Knuesel, Irene; Yee, Benjamin K

    2009-10-01

    The synaptic Ras/Rap-GTPase-activating protein (SynGAP) regulates specific intracellular events following N-methyl-d-aspartate receptor (NMDAR) activation. Here, the impact of SynGAP heterozygous knockout (SG+/-) on NMDAR-dependent functions was assessed using different positive reinforcement schedules in instrumental conditioning. The knockout did not affect the temporal control of operant responding under a fixed interval (FI) schedule, but led to a putative enhancement in response vigor and/or disinhibition. When examined on differential reinforcement of low rates of response (DRL) schedules, SG+/- mice showed increased responding under DRL-4s and DRL-8s, without impairing the response efficiency (total rewards/total lever presses) because both rewarded and nonrewarded presses were elevated. Motivation was unaffected as evaluated using a progressive ratio (PR) schedule. Yet, SG+/- mice persisted in responding during extinction at the end of PR training, although an equivalent phenotype was not evident in extinction learning following FI-20s training. This extinction phenotype is therefore schedule-specific and cannot be generalized to Pavlovian conditioning. In conclusion, constitutive SynGAP reduction increases vigor in the execution of learned operant behavior without compromising its temporal control, yielding effects readily distinguishable from NMDAR blockade. PMID:19824778

  4. [From alcohol to liquid ecstasy (GHB)--a survey of old and modern knockout agents. Part 1: historic and classic knockout agents].

    PubMed

    Schütz, Harald; Jansen, Malin; Dettmeyer, Reinhard; Verhoff, Marcel A

    2011-01-01

    Alcohol has been the most important knockout drug in history and literature and continues to play an essential role up to now. Blunt force to the head in the form of a knockout punch is another mechanism leading to a transient loss of consciousness. Diethyl ether and chloroform are among the classical knockout substances. Although they have meanwhile been replaced by modern sedatives and hypnotics, their use is still observed in isolated cases. PMID:22039694

  5. The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study.

    PubMed

    Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

    2016-09-01

    Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. PMID:27453341

  6. Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica.

    PubMed

    Liu, Hailong; Han, Jing; Liu, Xiaoqing; Zhou, Jian; Xiang, Hua

    2011-06-20

    The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (ΔpyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ΔpyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea. PMID:21703550

  7. Pauli blocking and medium effects in nucleon knockout reactions

    SciTech Connect

    Bertulani, C. A.; De Conti, C.

    2010-06-15

    We study medium modifications of the nucleon-nucleon (NN) cross sections and their influence on the nucleon knockout reactions. Using the eikonal approximation, we compare the results obtained with free NN cross sections with those obtained with a purely geometrical treatment of Pauli blocking and with NN obtained with more elaborated Dirac-Bruecker methods. The medium effects are parametrized in terms of the baryon density. We focus on symmetric nuclear matter, although the geometrical Pauli blocking also allows for the treatment of asymmetric nuclear matter. It is shown that medium effects can change the nucleon knockout cross sections and momentum distributions up to 10% in the energy range E{sub lab}=50-300 MeV/nucleon. The effect is more evident in reactions involving halo nuclei.

  8. Knockout driven reactions in complex molecules and their clusters

    NASA Astrophysics Data System (ADS)

    Gatchell, Michael; Zettergren, Henning

    2016-08-01

    Energetic ions lose some of their kinetic energy when interacting with electrons or nuclei in matter. Here, we discuss combined experimental and theoretical studies on such impulse driven reactions in polycyclic aromatic hydrocarbons (PAHs), fullerenes, and pure or mixed clusters of these molecules. These studies show that the nature of excitation is important for how complex molecular systems respond to ion/atom impact. Rutherford-like nuclear scattering processes may lead to prompt atom knockout and formation of highly reactive fragments, while heating of the molecular electron clouds in general lead to formation of more stable and less reactive fragments. In this topical review, we focus on recent studies of knockout driven reactions, and present new calculations of the angular dependent threshold (displacement) energies for such processes in PAHs. The so-formed fragments may efficiently form covalent bonds with neighboring molecules in clusters. These unique molecular growth processes may be important in astrophysical environments such as low velocity shock waves.

  9. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    PubMed Central

    2014-01-01

    Glycosaminoglycans (GAGs) are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs. PMID:25126564

  10. Creation and Preliminary Characterization of a Leptin Knockout Rat

    PubMed Central

    Vaira, Sergio; Yang, Chang; McCoy, Aaron; Keys, Kelly; Xue, Shurong; Weinstein, Edward J.; Novack, Deborah V.

    2012-01-01

    Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses. PMID:22948215

  11. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  12. Targeted gene knockout in chickens mediated by TALENs

    PubMed Central

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-01-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  13. Murine mentors: transgenic and knockout models of surgical disease.

    PubMed Central

    Arbeit, J M; Hirose, R

    1999-01-01

    OBJECTIVE: Transgenic and knockout technologies have emerged from the "molecular biology revolution" as unprecedented techniques for manipulating gene function in intact mice. The goals of this review are to outline the techniques of creating transgenic and knockout mice, and to demonstrate their use in elucidation of the molecular mechanisms underlying common surgical diseases. SUMMARY BACKGROUND DATA: Gain of gene function is created by transgenic technology, whereas gene function is ablated using gene knockouts. Each technique has distinctive applications and drawbacks. A unique feature of genetically manipulated mice is that combinatorial genetic experiments can be executed that precisely define the functional contribution of a gene to disease progression. Transgenic and knockout mouse models of wound healing, cardiovascular disease, transplant immunology, gut motility and inflammatory bowel disease, and oncology are beginning to illuminate the precise molecular regulation of these diseases. Transgenic technology has also been extended to larger mammals such as pigs, with the goal of using genetic manipulation of the xenogenic immune response to increase the availability of transplant organs. Continual refinements in gene manipulation technology in mice offer the opportunity to turn genes on or off at precise time intervals and in particular tissues, according to the needs of the investigator. Ultimately, investigation of disease development and progression in genetically manipulated mammals may delineate new molecular targets for drug discovery and provide novel platforms for drug efficacy screens. CONCLUSIONS: Emulation of human disease and therapy using genetically manipulated mammals fulfills a promise of molecular medicine: fusion of molecular biochemistry with "classical" biology and physiology. Surgeons have unique skills spanning both worlds that can facilitate their success in this expanding arena. PMID:9923797

  14. Generation of Gene Knockout Mice by ES Cell Microinjection

    PubMed Central

    Longenecker, Glenn; Kulkarni, Ashok B

    2009-01-01

    This unit lists and describes protocols used in the production of chimeric mice leading to the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols in the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipettes, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID:19731226

  15. Effect of arylamine acetyltransferase Nat3 gene knockout on N-acetylation in the mouse.

    PubMed

    Sugamori, K S; Brenneman, D; Wong, S; Gaedigk, A; Yu, V; Abramovici, H; Rozmahel, R; Grant, D M

    2007-07-01

    Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(-/-) knockout strain to further investigate the functional role of Nat3. Nat3(-/-) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(-/-) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(-/-) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(-/-) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(-/-) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(-/-) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities. PMID:17403913

  16. The evolution of thymic lymphomas in p53 knockout mice.

    PubMed

    Dudgeon, Crissy; Chan, Chang; Kang, Wenfeng; Sun, Yvonne; Emerson, Ryan; Robins, Harlan; Levine, Arnold J

    2014-12-01

    Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors' driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas. PMID:25452272

  17. Knock-out models reveal new aquaporin functions.

    PubMed

    Verkman, Alan S

    2009-01-01

    Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ' aquaglyceroporins', aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function - as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer. PMID:19096787

  18. Monocular Elevation Deficiency - Double Elevator Palsy

    MedlinePlus

    ... Español Condiciones Chinese Conditions Monocular Elevation Deficiency/ Double Elevator Palsy En Español Read in Chinese What is monocular elevation deficiency (Double Elevator Palsy)? Monocular Elevation Deficiency, also known by the ...

  19. Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

    PubMed

    Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

    2016-02-15

    Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions. PMID:26857489

  20. Epithelial-specific knockout of the Rac1 gene leads to enamel defects

    PubMed Central

    Huang, Zhan; Kim, Jieun; Lacruz, Rodrigo; Bringas, Pablo; Kaartinen, Vesa M.; Snead, Malcolm L.

    2015-01-01

    Rac1 encodes a 21kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction; activities with consequences to cell growth, adhesion, migration, and differentiation. To examine the role(s) played by Rac1 protein in cell-to-matrix interaction and in enamel matrix biomineralization we used the Cre/loxP binary recombination system to characterize enamel matrix proteins expression and enamel formation in Rac1 knockout mice. Mating between mice bearing the floxed Rac1 allele with mice bearing a keratin14-Cre transgene generate animals in which Rac1 is absent from epithelial organs. The enamel of Rac1 conditional knockout mouse was characterized by computerized tomography (microCT), light microscopy, histochemistry, and back-scatter electron microscopy. Enamel matrix protein expression was analyzed by Western blotting. Major findings showed that the Tomes’ processes of Rac1−/− ameloblasts loose contact with the forming enamel matrix in un-erupted teeth. The abundance of amelogenin and ameloblastin was reduced in the Rac1−/− ameloblasts. After eruption, the enamel from the Rac1−/− mice displayed severe structural defects with the complete loss of enamel. These results support an essential role for Rac1 function in the dental epithelium involving cell-matrix interaction and matrix biomineralization. PMID:22243243

  1. Double layers without current

    SciTech Connect

    Perkins, F.W.; Sun, Y.C.

    1980-11-01

    The steady-state solution of the nonlinear Vlasov-Poisson equations is reduced to a nonlinear eigenvalue problem for the case of double-layer (potential drop) boundary conditions. Solutions with no relative electron-ion drifts are found. The kinetic stability is discussed. Suggestions for creating these states in experiments and computer simulations are offered.

  2. An IPTG Inducible Conditional Expression System for Mycobacteria

    PubMed Central

    Ravishankar, Sudha; Ambady, Anisha; Ramu, Haripriya; Mudugal, Naina Vinay; Tunduguru, Ragadeepthi; Anbarasu, Anand; Sharma, Umender K.; Sambandamurthy, Vasan K.; Ramaiah, Sudha

    2015-01-01

    Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression

  3. An IPTG Inducible Conditional Expression System for Mycobacteria.

    PubMed

    Ravishankar, Sudha; Ambady, Anisha; Ramu, Haripriya; Mudugal, Naina Vinay; Tunduguru, Ragadeepthi; Anbarasu, Anand; Sharma, Umender K; Sambandamurthy, Vasan K; Ramaiah, Sudha

    2015-01-01

    Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression

  4. Double layers and electrostatic shocks

    NASA Technical Reports Server (NTRS)

    Hershkowitz, N.

    1981-01-01

    It is shown that it is useful to define double layers and shocks so that the ion phase spaces of double layers are the mirror image (about zero ion velocity) of the ion phase spaces for laminar electrostatic shocks. The distinguishing feature is the direction of the free ion velocity. It is also shown that double layers can exist without the presence of trapped ions. The Bohm condition for double layers, that the ion drift velocity on the high potential side must be greater than the ion sound velocity, is shown to be related to a requirement of a lower limit on the Mach number of laminar electrostatic shocks

  5. Outer-shell double photoionization of CH2Cl2

    NASA Astrophysics Data System (ADS)

    Alcantara, K. F.; Gomes, A. H. A.; Wolff, W.; Sigaud, L.; Santos, A. C. F.

    2014-01-01

    In this work the roles of the shake-off and knockout processes in the double photoionization of the CH2Cl2 molecule have been studied. The probabilities for both mechanisms accompanying valence-shell photoionization have been estimated as a function of incident photon energy using Samson's (1990) [5] and Thomas's (1994) [3] models, respectively. The experimental results are in qualitative accord with the models.

  6. Pre-Equilibrium Cluster Emission with Pickup and Knockout

    SciTech Connect

    Betak, E.

    2005-05-24

    We present a generalization of the Iwamoto-Harada-Bisplinghoff pre-equilibrium model of light cluster formation and emission, which is enhanced by allowing for possible admixtures of knockout for strongly coupled ejectiles, like {alpha}'s. The model is able to attain the Weisskopf-Ewing formula for compound-nucleus decay at long-time limit; it keeps the philosophy of pre-equilibrium decay during the equilibration stage and it describes the initial phase of a reaction as direct process(es) expressed using the language of the exciton model.

  7. Loss of oocytes due to conditional ablation of Murine double minute 2 (Mdm2) gene is p53-dependent and results in female sterility.

    PubMed

    Livera, Gabriel; Uzbekov, Rustem; Jarrier, Peggy; Fouchécourt, Sophie; Duquenne, Clotilde; Parent, Anne-Simone; Marine, Jean-Christophe; Monget, Philippe

    2016-08-01

    Murine double minute 2 and 4 (Mdm2, Mdm4) are major p53-negative regulators, preventing thus uncontrolled apoptosis induction in numerous cell types, although their function in the female germ line has received little attention. In the present work, we have generated mice with specific invalidation of Mdm2 and Mdm4 genes in the mouse oocyte (Mdm2(Ocko) and Mdm4(Ocko) mice), to test their implication in survival of these germ cells. Most of the Mdm2(Ocko) but not Mdm4(Ocko) mice were sterile, with a dramatic reduction of the weight of ovaries and genital tract, a strong increase in follicle-stimulating hormone and luteinizing hormone serum levels, and a reduction of anti-mullerian hormone serum levels. Histological analyses revealed an obvious decrease of the number of growing follicles beyond the primary stage in Mdm2(Ocko) ovaries in comparison to controls, with a pronounced increase in the apparition of primary atretic follicles, most being devoid of oocyte. Similar phenotypes were observed with Mdm2(Ocko) Mdm4(Ocko) ovaries, with no worsening of the phenotype. However, we failed to detect any increase in p53 level in mutant oocytes, nor any other apoptotic marker, introgression of this targeted invalidation in p53-/- mice restored the fertility of females. This study is the first to show that Mdm2, but not Mdm4, has a critical role in oocyte survival and would be involved in premature ovarian insufficiency phenotype. PMID:27364741

  8. Fenoverine: smooth muscle synchronizer for the management of gastro-intestinal conditions. II. A trimebutine-controlled, double-blind, crossover clinical evaluation.

    PubMed

    Camarri, E

    1986-01-01

    A double-blind, crossover trial was carried out in 40 in-patients with gastro-intestinal spasmodic syndromes to compare the effectiveness and tolerance of fenoverine and trimebutine. Patients were allocated at random to receive either 100 mg fenoverine or 150 mg trimebutine 3-times daily for 20 days and were then crossed over, without a wash-out period, to the alternative medication for a further 20 days. After the first dose, pain severity was monitored over 4 hours and changes in intensity compared between groups. During the two 20-day periods, the proportion of patients in complete or almost complete remission was monitored at 10-day intervals, and the pooled data similarly compared. At the end of the 40-day trial period, patients stated their preference for one or other treatment, and the relevant data were processed by sequential analysis. Subjective signs of adverse effects were monitored by questioning every 10 days, and haematology and haematochemistry before and after each phase of the study. The results showed that fenoverine produced significantly greater pain relief after a single dose in comparison with trimebutine over the 4 hours of observation. Similarly, it gave significantly more favourable clinical results after both the 10th and 20th day of treatment. Finally, according to the patients' preference, fenoverine was significantly preferred (p less than 0.05) in comparison with trimebutine. Neither treatment was associated with the onset of signs of possible adverse reactions, either subjective or objective. PMID:3516580

  9. Toll-like receptor 4 knockout ameliorates neuroinflammation due to lung-brain interaction in mechanically ventilated mice.

    PubMed

    Chen, Ting; Chen, Chang; Zhang, Zongze; Zou, Yufeng; Peng, Mian; Wang, Yanlin

    2016-08-01

    Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system, and increasing evidence supports its role in inflammation, stress, and tissue injury, including injury to the lung and brain. We aimed to investigate the effects of TLR4 on neuroinflammation due to the lung-brain interaction in mechanically ventilated mice. Male wild-type (WT) C57BL/6 and TLR4 knockout (TLR4 KO) mice were divided into three groups: (1) control group (C): spontaneous breathing; (2) anesthesia group (A): spontaneous breathing under anesthesia; and (3) mechanical ventilation group (MV): 6h of MV under anesthesia. The behavioral responses of mice were tested with fear conditioning tests. The histological changes in the lung and brain were assessed using hematoxylin-eosin (HE) staining. The level of TLR4 mRNA in tissue was measured using reverse transcription-polymerase chain reaction (RT-PCR). The levels of inflammatory cytokines were measured with an enzyme-linked immunosorbent assay (ELISA). Microgliosis, astrocytosis, and the TLR4 immunoreactivity in the hippocampus were measured by double immunofluorescence. MV mice exhibited impaired cognition, and this impairment was less severe in TLR4 KO mice than in WT mice. In WT mice, MV increased TLR4 mRNA expression in the lung and brain. MV induced mild lung injury, which was prevented in TLR4 KO mice. MV mice exhibited increased levels of inflammatory cytokines, increased microglia and astrocyte activation. Microgliosis was alleviated in TLR4 KO mice. MV mice exhibited increased TLR4 immunoreactivity, which was expressed in microglia and astrocytes. These results demonstrate that TLR4 is involved in neuroinflammation due to the lung-brain interaction and that TLR4 KO ameliorates neuroinflammation due to lung-brain interaction after prolonged MV. In addition, Administration of a TLR4 antagonist (100μg/mice) to WT mice also significantly attenuated neuroinflammation of lung-brain interaction due to prolonged MV. TLR4 antagonism

  10. Double Layers in Astrophysics

    NASA Technical Reports Server (NTRS)

    Williams, Alton C. (Editor); Moorehead, Tauna W. (Editor)

    1987-01-01

    Topics addressed include: laboratory double layers; ion-acoustic double layers; pumping potential wells; ion phase-space vortices; weak double layers; electric fields and double layers in plasmas; auroral double layers; double layer formation in a plasma; beamed emission from gamma-ray burst source; double layers and extragalactic jets; and electric potential between plasma sheet clouds.

  11. Generation of myometrium-specific Bmal1 knockout mice for parturition analysis.

    PubMed

    Ratajczak, Christine K; Asada, Minoru; Allen, Gregg C; McMahon, Douglas G; Muglia, Lisa M; Smith, Donté; Bhattacharyya, Sandip; Muglia, Louis J

    2012-01-01

    Human and rodent studies indicate a role for circadian rhythmicity and associated clock gene expression in supporting normal parturition. The importance of clock gene expression in tissues besides the suprachiasmatic nucleus is emerging. Here, a Bmal1 conditional knockout mouse line and a novel Cre transgenic mouse line were used to examine the role of myometrial Bmal1 in parturition. Ninety-two percent (22/24) of control females but only 64% (14/22) of females with disrupted myometrial Bmal1 completed parturition during the expected time window of 5p.m. on Day 19 through to 9a.m. on Day 19.5 of gestation. However, neither serum progesterone levels nor uterine transcript expression of the contractile-associated proteins Connexin43 and Oxytocin receptor differed between females with disrupted myometrial Bmal1 and controls during late gestation. The data indicate a role for myometrial Bmal1 in maintaining normal time of day of parturition. PMID:22697126

  12. Combination of cell culture assays and knockout mouse analyses for the study of opioid partial agonism.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Sora, Ichiro; Ikeda, Kazutaka

    2010-01-01

    Nonselective opioid partial agonists, such as buprenorphine, butorphanol, and pentazocine, have been widely used as analgesics and for anti-addiction therapy. However, the precise molecular mechanisms underlying the therapeutic and rewarding effects of these drugs have not been clearly delineated. Recent success in developing mu-opioid receptor knockout (MOP-KO) mice has elucidated the molecular mechanisms underlying the effects of morphine and other opioids. We have revealed the in vivo roles of MOPs in the effects of opioid partial agonists by using MOP-KO mice for behavioral tests (e.g., several kinds of antinociceptive tests for analgesic effects, conditioned place preference test for dependence). The combination of the cell culture assays using cDNA for mu, delta, and kappa opioid receptors and the behavioral tests using MOP-KO mice has provided novel theories on the molecular mechanisms underlying the effects of opioid ligands, especially opioid partial agonists. PMID:20336435

  13. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.

    PubMed

    Grav, Lise Marie; Lee, Jae Seong; Gerling, Signe; Kallehauge, Thomas Beuchert; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup

    2015-09-01

    The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further. PMID:25864574

  14. Double screening

    NASA Astrophysics Data System (ADS)

    Gratia, Pierre; Hu, Wayne; Joyce, Austin; Ribeiro, Raquel H.

    2016-06-01

    Attempts to modify gravity in the infrared typically require a screening mechanism to ensure consistency with local tests of gravity. These screening mechanisms fit into three broad classes; we investigate theories which are capable of exhibiting more than one type of screening. Specifically, we focus on a simple model which exhibits both Vainshtein and kinetic screening. We point out that due to the two characteristic length scales in the problem, the type of screening that dominates depends on the mass of the sourcing object, allowing for different phenomenology at different scales. We consider embedding this double screening phenomenology in a broader cosmological scenario and show that the simplest examples that exhibit double screening are radiatively stable.

  15. Studying TGF-beta superfamily signaling by knockouts and knockins.

    PubMed

    Chang, H; Lau, A L; Matzuk, M M

    2001-06-30

    The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues. PMID:11451570

  16. Screening methods to identify TALEN-mediated knockout mice.

    PubMed

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms. PMID:24521866

  17. Creation and preliminary characterization of a Tp53 knockout rat

    PubMed Central

    McCoy, Aaron; Besch-Williford, Cynthia L.; Franklin, Craig L.; Weinstein, Edward J.; Cui, Xiaoxia

    2013-01-01

    SUMMARY The tumor suppressor TP53 plays a crucial role in cancer biology, and the TP53 gene is the most mutated gene in human cancer. Trp53 knockout mouse models have been widely used in cancer etiology studies and in search for a cure of cancer with some limitations that other model organisms might help overcome. Via pronuclear microinjection of zinc finger nucleases (ZFNs), we created a Tp53 knockout rat that contains an 11-bp deletion in exon 3, resulting in a frameshift and premature terminations in the open reading frame. In cohorts of 25 homozygous (Tp53Δ11/Δ11), 37 heterozygous (Tp53Δ11/+) and 30 wild-type rats, the Tp53Δ11/Δ11 rats lived an average of 126 days before death or removal from study because of clinical signs of abnormality or formation of tumors. Half of Tp53Δ11/+ were removed from study by 1 year of age because of tumor formation. Both Tp53Δ11/+ and Tp53Δ11/Δ11 rats developed a wide spectrum of tumors, most commonly sarcomas. Interestingly, there was a strikingly high incidence of brain lesions, especially in Tp53Δ11/Δ11 animals. We believe that this mutant rat line will be useful in studying cancer types rarely observed in mice and in carcinogenicity assays for drug development. PMID:22917926

  18. Screening Methods to Identify TALEN-Mediated Knockout Mice

    PubMed Central

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms. PMID:24521866

  19. Norepinephrine Transporter Heterozygous Knockout Mice Exhibit Altered Transport and Behavior

    PubMed Central

    Fentress, HM; Klar, R; Krueger, JK; Sabb, T; Redmon, SN; Wallace, NM; Shirey-Rice, JK; Hahn, MK

    2013-01-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically-driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET+/−), demonstrating that they display an ~50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity, assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET+/− mouse establishes an activated state of existing, surface NET proteins. NET+/− mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris Water Maze. These data suggest recovery of near basal activity in NET+/− mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET+/− mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

  20. RAG1/2 knockout pigs with severe combined immunodeficiency.

    PubMed

    Huang, Jiao; Guo, Xiaogang; Fan, Nana; Song, Jun; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Zhao, Yu; Yan, Quanmei; Yi, Xiaoling; Schambach, Axel; Frampton, Jon; Esteban, Miguel A; Yang, Dongshan; Yang, Huaqiang; Lai, Liangxue

    2014-08-01

    Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research. PMID:24973446

  1. Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior.

    PubMed

    Fentress, H M; Klar, R; Krueger, J J; Sabb, T; Redmon, S N; Wallace, N M; Shirey-Rice, J K; Hahn, M K

    2013-11-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET(+/-) ), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET(+/-) mouse establishes an activated state of existing surface NET proteins. The NET(+/-) mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET(+/-) mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET(+/-) mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

  2. Study of 19C by One-Neutron Knockout

    NASA Astrophysics Data System (ADS)

    Hwang, Jongwon; Kim, Sunji; Satou, Yoshiteru; Orr, Nigel A.; Nakamura, Takashi; Kondo, Yosuke; Gibelin, Julien; Achouri, N. Lynda; Aumann, Thomas; Baba, Hidetada; Delaunay, Franck; Doornenbal, Pieter; Fukuda, Naoki; Inabe, Naohito; Isobe, Tadaaki; Kameda, Daisuke; Kanno, Daiki; Kobayashi, Nobuyuki; Kobayashi, Toshio; Kubo, Toshiyuki; Leblond, Sylvain; Lee, Jenny; Marqués, F. Miguel; Minakata, Ryogo; Motobayashi, Tohru; Murai, Daichi; Murakami, Tetsuya; Muto, Kotomi; Nakashima, Tomohiro; Nakatsuka, Noritsugu; Navin, Alahari; Nishi, Seijiro; Ogoshi, Shun; Otsu, Hideaki; Sato, Hiromi; Shimizu, Yohei; Suzuki, Hiroshi; Takahashi, Kento; Takeda, Hiroyuki; Takeuchi, Satoshi; Tanaka, Ryuki; Togano, Yasuhiro; Tuff, Adam G.; Vandebrouck, Marine; Yoneda, Ken-ichiro

    2016-03-01

    The spectroscopic structure of 19C, a prominent one-neutron halo nucleus, has been studied with a 20C secondary beam at 290 MeV/nucleon and a carbon target. Neutron-unbound states populated by the one-neutron knockout reaction were investigated by means of the invariant mass method. The preliminary relative energy spectrum and parallel momentum distribution of the knockout residue, 19C*, were reconstructed from the measured four momenta of the 18C fragment, neutron, and beam. Three resonances were observed in the spectrum, which correspond to the states at Ex = 0.62(9), 1.42(10), and 2.89(10) MeV. The parallel momentum distributions for the 0.62-MeV and 2.89-MeV states suggest spin-parity assignments of 5/2+ and 1/2-, respectively. The 1.42-MeV state is in line with the reported 5/22+ state.

  3. Enhanced Susceptibility of Ago1/3 Double-Null Mice to Influenza A Virus Infection

    PubMed Central

    Van Stry, Melanie; Oguin, Thomas H.; Cheloufi, Sihem; Vogel, Peter; Watanabe, Makiko; Pillai, Meenu R.; Dash, Pradyot; Thomas, Paul G.; Hannon, Gregory J.

    2012-01-01

    RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation. PMID:22318144

  4. Enhanced susceptibility of Ago1/3 double-null mice to influenza A virus infection.

    PubMed

    Van Stry, Melanie; Oguin, Thomas H; Cheloufi, Sihem; Vogel, Peter; Watanabe, Makiko; Pillai, Meenu R; Dash, Pradyot; Thomas, Paul G; Hannon, Gregory J; Bix, Mark

    2012-04-01

    RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation. PMID:22318144

  5. Caveolin-1 gene knockout impairs nitrergic function in mouse small intestine

    PubMed Central

    El-Yazbi, Ahmed F; Cho, Woo-Jung; Boddy, Geoffrey; Daniel, Edwin E

    2005-01-01

    Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1−/−) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1−/− mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1−/− mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10−4 M), but relaxation of Cav1−/− animals was affected much less. Also, Cav1−/− mice tissues showed reduced relaxation responses to sodium nitroprusside (100 μM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3′ : 5′-cyclic monophosphate (100 μM). Apamin (10−6 M) significantly reduced relaxations to EFS in NANC conditions in Cav1−/− mice, but not in controls. The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators. PMID:15937515

  6. Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors

    PubMed Central

    Park, Ryan J.; Shen, Hongying; Liu, Lijuan; Liu, Xinran; Ferguson, Shawn M.; De Camilli, Pietro

    2013-01-01

    Summary Dynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors. PMID:24046449

  7. Determination of Hypersensitivity to Genotoxic Agents Among Escherichia coli Single Gene Knockout Mutants

    PubMed Central

    Becket, Elinne; Chen, Frank; Tamae, Cindy; Miller, Jeffrey H.

    2010-01-01

    Summary We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2’-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct “sensitivity profile” that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB. PMID:20674514

  8. Determination of hypersensitivity to genotoxic agents among Escherichia coli single gene knockout mutants.

    PubMed

    Becket, Elinne; Chen, Frank; Tamae, Cindy; Miller, Jeffrey H

    2010-09-01

    We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2'-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct "sensitivity profile" that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB. PMID:20674514

  9. Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

    PubMed Central

    Wargelius, Anna; Leininger, Sven; Skaftnesmo, Kai Ove; Kleppe, Lene; Andersson, Eva; Taranger, Geir Lasse; Schulz, Rüdiger W; Edvardsen, Rolf B

    2016-01-01

    Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish. PMID:26888627

  10. Study on the d state of platinum in Pt/SiO sub 2 and Na/Pt/SiO sub 2 catalysts under C double bond C hydrogenation conditions by X-ray absorption near-edge structure spectroscopy

    SciTech Connect

    Yoshitake, Hideaki; Iwasawa, Yasuhiro )

    1991-09-19

    The change in the d-electron density of platinum during D{sub 2} + CH{sub 2}{double bond}CHX reactions on Pt/SiO{sub 2} and Na/Pt/SiO{sub 2} catalysts and its influence on the catalysis were studied by X-ray absorption near-edge structure (XANES) spectroscopy, kinetics and FT-IR. It was demonstrated from the change of the white lines in XANES spectra at Pt L{sub 2} and L{sub 3} edges that CH{sub 2}{double bond}CHX (X = H, CH{sub 3}, COCH{sub 3}, CF{sub 3}, and CN) is adsorbed on the Pt surface and extracts the electrons of the d state. Hence, the deuterogenation rate is reduced as the value of Hammett's {sigma}{sub P} increases. The linear free energy relationship between the reaction rate and {sigma}{sub P} was observed for the deuterogenation of CH{sub 2}{double bond}CHX. The rate of ethene deuterogenation was promoted by Na{sub 2}O addition. The electron density of unoccupied d states of pt under vacuum decreased by Na{sub 2}O addition, indicating the electron donation from Na{sub 2}O addition. The electron density of unoccupied d states of Pt under vacuum decreased by Na{sub 2}O addition, indicating the electron donation from Na{sub 2}O addition. However, most of these additional electrons were observed to move to ethene under reaction conditions. The acceptor of the electrons was suggested by di-{sigma}-ethene by the shift of {upsilon}(C-H). The kinetic parameters are discussed in relation to the change in the d state of Pt as a function of {sigma}{sub P} and Na quantity.

  11. Kidney development and gene expression in the HIF2alpha knockout mouse.

    PubMed

    Steenhard, Brooke M; Freeburg, Paul B; Isom, Kathryn; Stroganova, Larysa; Borza, Dorin-Bogdan; Hudson, Billy G; St John, Patricia L; Zelenchuk, Adrian; Abrahamson, Dale R

    2007-04-01

    The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular

  12. The Effect of Different Photoperiods in Circadian Rhythms of Per3 Knockout Mice

    PubMed Central

    Pereira, D. S.; van der Veen, D. R.; Gonçalves, B. S. B.; Tufik, S.; von Schantz, M.; Archer, S. N.; Pedrazzoli, M.

    2014-01-01

    The aim of this study was to analyse the circadian behavioural responses of mice carrying a functional knockout of the Per3 gene (Per3−/−) to different light : dark (L : D) cycles. Male adult wild-type (WT) and Per3−/− mice were kept under 12-hour light : 12-hour dark conditions (12L : 12D) and then transferred to either a short or long photoperiod and subsequently released into total darkness. All mice were exposed to both conditions, and behavioural activity data were acquired through running wheel activity and analysed for circadian characteristics during these conditions. We observed that, during the transition from 12L : 12D to 16L : 8D, Per3−/− mice take approximately one additional day to synchronise to the new L : D cycle compared to WT mice. Under these long photoperiod conditions, Per3−/− mice were more active in the light phase. Our results suggest that Per3−/− mice are less sensitive to light. The data presented here provides further evidence that Per3 is involved in the suppression of behavioural activity in direct response to light. PMID:24982860

  13. Knockout of Foxp2 disrupts vocal development in mice.

    PubMed

    Castellucci, Gregg A; McGinley, Matthew J; McCormick, David A

    2016-01-01

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control. PMID:26980647

  14. Drop tests of the Three Mile Island knockout canister

    SciTech Connect

    Box, W D; Aaron, W S; Shappert, L B; Childress, P C; Quinn, G J; Smith, J V

    1987-01-01

    A type of Three Mile Island Unit 2 (TMI-2) defueling canister, called a ''knockout'' canister, was subjected to a series of drop tests at the Oak Ridge National Laboratory's Drop Test Facility. These tests confirmed the structural integrity of internal fixed neutron poisons in support of a request for NRC licensing of this type of canister for the shipment of TMI-2 reactor fuel debris to the Idaho National Engineering Laboratory (INEL) for the Core Examination R and D Program. This report presents the data generated and the results obtained from a series of four drop tests that included two drops with the test assembly in the vertical position and two drops with the assembly in the horizontal position.

  15. Progranulin Knockout Accelerates Intervertebral Disc Degeneration in Aging Mice

    PubMed Central

    Zhao, Yun-peng; Tian, Qing-yun; Liu, Ben; Cuellar, Jason; Richbourgh, Brendon; Jia, Tang-hong; Liu, Chuan-ju

    2015-01-01

    Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases. PMID:25777988

  16. Nonsudden limits of heavy-ion induced knockout reactions.

    PubMed

    Flavigny, F; Obertelli, A; Bonaccorso, A; Grinyer, G F; Louchart, C; Nalpas, L; Signoracci, A

    2012-06-22

    We report on the single neutron and proton removal reactions from unstable nuclei with large asymmetry ΔS = S(n)-S(p) at incident energies below 80 MeV/nucleon. Strong nonsudden effects are observed in the case of deeply-bound-nucleon removal. The corresponding parallel momentum distributions exhibit an abrupt cutoff at high momentum that corresponds to an energy threshold occurring when the incident energy per particle is of comparable magnitude to the nucleon separation energy. A large low-momentum tail is related to both dissipative processes and the dynamics of the nucleon removal process. New limits for the applicability of the sudden and eikonal approximations in nucleon knockout are given. PMID:23004591

  17. Spectroscopy of 17C via one-neutron knockout reaction

    NASA Astrophysics Data System (ADS)

    Kim, Sunji; Hwang, Jongwon; Satou, Yoshiteru; Orr, Nigel A.; Nakamura, Takashi; Kondo, Yosuke; Gibelin, Julien; Achouri, N. Lynda; Aumann, Thomas; Baba, Hidetada; Delaunay, Franck; Doornenbal, Pieter; Fukuda, Naoki; Inabe, Naohito; Isobe, Tadaaki; Kameda, Daisuke; Kanno, Daiki; Kobayashi, Nobuyuki; Kobayashi, Toshio; Kubo, Toshiyuki; Leblond, Sylvain; Lee, Jenny; Marqués, F. Miguel; Minakata, Ryogo; Motobayashi, Tohru; Murai, Daichi; Murakami, Tetsuya; Muto, Kotomi; Nakashima, Tomohiro; Nakatsuka, Noritsugu; Navin, Alahari; Nishi, Seijiro; Ogoshi, Shun; Otsu, Hideaki; Sato, Hiromi; Shimizu, Yohei; Suzuki, Hiroshi; Takahashi, Kento; Takeda, Hiroyuki; Takeuchi, Satoshi; Tanaka, Ryuki; Togano, Yasuhiro; Tuff, Adam G.; Vandebrouck, Marine; Yoneda, Ken-ichiro

    2016-03-01

    A spectroscopic study of 17C was performed via the one-neutron knockout reaction of 18C on a carbon target at RIKEN-RIBF. Three unbound states at excitation energies of 2.66(2), 3.16(5), and 3.97(3) MeV (preliminary) were observed. The energies are compared with shell-model calculations and existing measurements to deduce their spin-parities. From the comparison, the states at 2.66(2) and 3.97(3) MeV are suggested to be 1/2- and 3/2-, respectively. From its decay property, the state at 3.16(5) MeV is indicated to be 9/2+.

  18. Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

    SciTech Connect

    Ezaki, Hisao; Yoshida, Yuichi; Saji, Yukiko; Takemura, Takayo; Fukushima, Juichi; Matsumoto, Hitoshi; Kamada, Yoshihiro; Wada, Akira; Igura, Takumi; Kihara, Shinji; Funahashi, Tohru; Shimomura, Iichiro; Tamura, Shinji; Kiso, Shinichi Hayashi, Norio

    2009-01-02

    We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) {alpha} and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.

  19. Fission product release from nuclear fuel by recoil and knockout

    NASA Astrophysics Data System (ADS)

    Lewis, B. J.

    1987-03-01

    An analytical model has been developed to describe the fission product release from nuclear fuel arising from the surface-fission release mechanisms of recoil and knockout. Release expressions are evaluated and compared to the short-lived activity measurements from in-reactor experiments with intact operating fuel. Recoil is shown to be an important process for releasing fission products from free UO 2 surfaces into the fuel-to-sheath gap. The model is also applied to tramp uranium in a power reactor primary heat transport circuit where it is demonstrated that recoil is the dominant release mechanism for small particles of fuel which are deposited on in-core surfaces. A methodology is established whereby release from surface contamination can be distinguished from that of fuel pin failure.

  20. CRISPR-Cas9 mediated gene knockout in primary human airway epithelial cells reveals a pro-inflammatory role for MUC18

    PubMed Central

    Chu, Hong Wei; Rios, Cydney; Huang, Chunjian; Wesolowska-Andersen, Agata; Burchard, Esteban G.; O'Connor, Brian P.; Fingerlin, Tasha E.; Nichols, David; Reynolds, Susan D.; Seibold, Max A.

    2015-01-01

    Targeted knockout of genes in primary human cells using CRISPR-Cas9 mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a pro-inflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9 mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a pro-inflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. PMID:26043872

  1. Bioelectric characterization of epithelia from neonatal CFTR knockout ferrets.

    PubMed

    Fisher, John T; Tyler, Scott R; Zhang, Yulong; Lee, Ben J; Liu, Xiaoming; Sun, Xingshen; Sui, Hongshu; Liang, Bo; Luo, Meihui; Xie, Weiliang; Yi, Yaling; Zhou, Weihong; Song, Yi; Keiser, Nicholas; Wang, Kai; de Jonge, Hugo R; Engelhardt, John F

    2013-11-01

    Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport. PMID:23782101

  2. Intercellular Adhesion Molecule 1 Knockout Abrogates Radiation Induced Pulmonary Inflammation

    NASA Astrophysics Data System (ADS)

    Hallahan, Dennis E.; Virudachalam, Subbulakshmi

    1997-06-01

    Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration

  3. Intercellular adhesion molecule 1 knockout abrogates radiation induced pulmonary inflammation.

    PubMed

    Hallahan, D E; Virudachalam, S

    1997-06-10

    Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration

  4. Bioelectric Characterization of Epithelia from Neonatal CFTR Knockout Ferrets

    PubMed Central

    Fisher, John T.; Tyler, Scott R.; Zhang, Yulong; Lee, Ben J.; Liu, Xiaoming; Sun, Xingshen; Sui, Hongshu; Liang, Bo; Luo, Meihui; Xie, Weiliang; Yi, Yaling; Zhou, Weihong; Song, Yi; Keiser, Nicholas; Wang, Kai; de Jonge, Hugo R.

    2013-01-01

    Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin–stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)–inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin–inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport. PMID:23782101

  5. Reduced Extinction of Hippocampal-Dependent Memories in CPEB Knockout Mice

    ERIC Educational Resources Information Center

    Zearfoss, N. Ruth; Richter, Joel D.; Berger-Sweeney, Joanne

    2006-01-01

    CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of…

  6. Comparison of Propofol and Ketamine-Propofol Mixture (Ketofol) on Laryngeal Tube-Suction II Conditions and Hemodynamics: A Randomized, Prospective, Double-Blind Trial☆

    PubMed Central

    Ozgul, Ulku; Begec, Zekine; Karahan, Kalender; Ali Erdogan, Mehmet; Said Aydogan, Mustafa; Colak, Cemil; Durmus, Mahmut; Ozcan Ersoy, M.

    2013-01-01

    Objective The aim of our study is to compare the effect of ketamine–propofol mixture (ketofol) and propofol on the laryngeal tube-Suction II (LTS II) insertion conditions and hemodynamics. Methods Eighty American Society of Anesthesiologists class 1 and 2 patients were divided into 2 random groups to receive either 1 µg/kg remifentanil and propofol 2 mg/kg in Group P (n = 40), or 1 µg/kg remifentanil and ketofol (using a 1:1 single syringe mixture of 5 mg/mL ketamine and 5 mg/mL propofol) in Group K (n = 40) before induction of anesthesia. After induction, LTS II was inserted. Heart rate and noninvasive blood pressure were recorded before induction of anesthesia (t0); immediately following induction (t1); immediately after LTS II insertion (t2); and 3 minutes (t3), 5 minutes (t4), and 10 (t5) minutes after LTS II insertion. Conditions of insertion of LTS II were assessed and scored 1 to 3 using 6 variables as follows: mouth opening, swallowing, coughing, head and body movements, laryngospasm, and ease of LTS II insertion by the same experienced anesthesiologist who did not know the agents. LTS II insertion summed score was prepared depending upon these variables. Results In regard to LTS II insertion summed score, Group K was more favorable than Group P (P < 0.05). Apnea duration was longer in Group P (385.0 seconds [range = 195.0–840.0 seconds]) compared with Group K (325.50 seconds [range = 60.0–840.0 seconds]) but this was not statically significant. The heart rate values were significantly lower at all measurement intervals in both groups compared with the baseline values (P < 0.05). There was no difference in heart rate between Group P and Group K. The mean arterial pressure values were significantly lower at all measurement intervals in Group P compared with baseline values (P < 0.05). In Group K, the mean arterial pressure values were significantly lower at all measurement intervals compared with the baseline values, except t2 (P < 0.05). There was a

  7. Health and population effects of rare gene knockouts in adult humans with related parents.

    PubMed

    Narasimhan, Vagheesh M; Hunt, Karen A; Mason, Dan; Baker, Christopher L; Karczewski, Konrad J; Barnes, Michael R; Barnett, Anthony H; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A; Giorda, Kristina; Griffiths, Christopher J; Hemingway, Harry; Jia, Zhilong; Kelly, M Ann; Khawaja, Hajrah A; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O'Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M; Tyler-Smith, Chris; Maher, Eamonn R; Trembath, Richard C; MacArthur, Daniel G; Wright, John; Durbin, Richard; van Heel, David A

    2016-04-22

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans. PMID:26940866

  8. Health and population effects of rare gene knockouts in adult humans with related parents

    PubMed Central

    Narasimhan, Vagheesh M.; Hunt, Karen A.; Mason, Dan; Baker, Christopher L.; Karczewski, Konrad J.; Barnes, Michael R.; Barnett, Anthony H.; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A.; Giorda, Kristina; Griffiths, Christopher J.; Hemingway, Harry; Jia, Zhilong; Kelly, M. Ann; Khawaja, Hajrah A.; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O’Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A.; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M.; Tyler-Smith, Chris; Maher, Eamonn R.; Trembath, Richard C.; MacArthur, Daniel G.; Wright, John; Durbin, Richard; van Heel, David A.

    2016-01-01

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localised away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans. PMID:26940866

  9. Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.

    PubMed

    Sampson, Kathleen E; Brinker, Amanda; Pratt, Jennifer; Venkatraman, Neetu; Xiao, Yongling; Blasberg, Jim; Steiner, Toni; Bourner, Maureen; Thompson, David C

    2015-02-01

    Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 μM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches. PMID:25388687

  10. Renal outer medullary potassium channel knockout models reveal thick ascending limb function and dysfunction.

    PubMed

    Wang, Tong

    2012-02-01

    The renal outer medullary potassium channel (ROMK) is an adenosine triphosphate-sensitive inward-rectifier potassium channel (Kir1.1 or KCNJ1) highly expressed in the cortical and medullary thick ascending limbs (TAL), connecting segment (CNT) and cortical collecting duct (CCD) in the mammalian kidney, where it serves to recycle potassium (K(+)) across the apical membrane in TAL and to secrete K(+) in the CNT and CCD. ROMK channel mutations cause type II Bartter's syndrome with salt wasting and dehydration, and ROMK knockout mice display a similar phenotype of Bartter's syndrome in humans. Studies from ROMK null mice indicate that ROMK is required to form both the small-conductance (30pS, SK) K channels and the 70pS (IK) K channels in the TAL. The availability of ROMK(-/-) mice has made it possible to study electrolyte transport along the nephron in order to understand the TAL function under physiological conditions and the compensatory mechanisms of salt and water transport under the conditions of TAL dysfunction. This review summarizes previous progress in the study of K(+) channel activity in the TAL and CCD, ion transporter expression and activities along the nephron, and renal functions under physiological and pathophysiological conditions using ROMK(-/-) mice. PMID:22038261

  11. Final-state interactions in two-nucleon knockout reactions

    NASA Astrophysics Data System (ADS)

    Colle, Camille; Cosyn, Wim; Ryckebusch, Jan

    2016-03-01

    Background: Exclusive two-nucleon knockout after electroexcitation of nuclei [A (e ,e'N N ) in brief] is considered to be a primary source of information about short-range correlations (SRCs) in nuclei. For a proper interpretation of the data, final-state interactions (FSIs) need to be theoretically controlled. Purpose: Our goal is to quantify the role of FSI effects in exclusive A (e ,e'p N ) reactions for four target nuclei representative of the whole mass region. Our focus is on processes that are SRC driven. We investigate the role of FSIs for two characteristic detector setups corresponding to "small" and "large" coverage of the available phase space. Method: Use is made of a factorized expression for the A (e ,e'p N ) cross section that is proportional to the two-body center-of-mass (c.m.) momentum distribution of close-proximity pairs. The A (e ,e'p p ) and A (e ,e'p n ) reactions for the target nuclei 12C,27Al,56Fe, and 208Pb are investigated. The elastic attenuation mechanisms in the FSIs are included using the relativistic multiple-scattering Glauber approximation (RMSGA). Single-charge exchange (SCX) reactions are also included. We introduce the nuclear transparency TAp N, defined as the ratio of exclusive (e ,e'p N ) cross sections on nuclei to those on "free" nucleon pairs, as a measure for the aggregated effect of FSIs in p N knockout reactions from nucleus A . A toy model is introduced in order to gain a better understanding of the A dependence of TAp N. Results: The transparency TAp N drops from 0.2 -0.3 for 12C to 0.04 -0.07 for 208Pb. For all considered kinematics, the mass dependence of TAp N can be captured by the power law TAp N∝A-λ with 0.4 ≲λ ≲0.5 . Apart from an overall reduction factor, we find that FSIs only modestly affect the distinct features of SRC-driven A (e ,e'p N ) which are dictated by the c.m. distribution of close-proximity pairs. Conclusion: The SCX mechanisms represent a relatively small (order of a few percent

  12. Complementation Test of Rpe65 Knockout and Tvrm148

    PubMed Central

    Wright, Charles B.; Chrenek, Micah A.; Foster, Stephanie L.; Duncan, Todd; Redmond, T. Michael; Pardue, Machelle T.; Boatright, Jeffrey H.; Nickerson, John M.

    2013-01-01

    Purpose. A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels. Methods. Normal phase HPLC was used to measure retinoid levels. Rpe65+/+, tvrm148/+ (T+/−), tvrm148/tvrm148 (T−/−), RPE65KO/KO (Rpe65−/−), and Rpe65T/− mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein. Results. The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T−/− or Rpe65−/− mice. Visual acuity in Rpe65+/+ and T+/− mouse was ∼0.382 c/d, but 0.037 c/d in T−/− mice at postnatal day 210 (P210). ERG response in T−/− mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T−/− mice was ∼70% of Rpe65+/+ by P210. Rpe65 mRNA levels in T−/− mice were unchanged, yet 14.5% of Rpe65+/+ protein levels was detected. Protein size was unchanged. Conclusions. A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65. PMID:23778877

  13. Developmental Divergence of Sleep-Wake Patterns in Orexin Knockout and Wild-Type Mice

    PubMed Central

    Coleman, Cassandra M.; Johnson, Eric D.; Shaw, Cynthia

    2008-01-01

    Narcolepsy, a disorder characterized by fragmented bouts of sleep and wakefulness during the day and night as well as cataplexy, has been linked in humans and non-human animals to the functional integrity of the orexinergic system. Adult orexin knockout mice and dogs with a mutation of the orexin receptor exhibit symptoms that mirror those seen in narcoleptic humans. As with narcolepsy, infant sleep-wake cycles in humans and rats are highly fragmented, with consolidated bouts of sleep and wakefulness developing gradually. Based on these common features of narcoleptics and infants, we hypothesized that the development of sleep-wake fragmentation in orexin knockout mice would be expressed as a developmental divergence between knockouts and wild-types, with the knockouts lagging behind the wild-types. We tested this hypothesis by recording the sleep-wake patterns of infant orexin knockout and wild-type mice across the first three postnatal weeks. Both knockouts and wild-types exhibited age-dependent, and therefore orexin-independent, quantitative and qualitative changes in sleep-wake patterning. At 3 weeks of age, however, by which time the sleep and wake bouts of the wild-types had consolidated further, the knockouts lagged behind the wild-types and exhibited significantly more bout fragmentation. These findings suggest the possibility that the fragmentation of behavioral states that characterizes narcolepsy in adults reflects reversion back toward the more fragmented sleep-wake patterns that characterize infancy. PMID:17284193

  14. Creatine transporter (CrT; Slc6a8) knockout mice as a model of human CrT deficiency.

    PubMed

    Skelton, Matthew R; Schaefer, Tori L; Graham, Devon L; Degrauw, Ton J; Clark, Joseph F; Williams, Michael T; Vorhees, Charles V

    2011-01-01

    Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2-4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT(⁻/y) (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT(⁻/y) mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT(⁻/y) mice showed increased average distance from the platform site. CrT(⁻/y) mice showed reduced novel object recognition and conditioned fear memory compared to CrT(+/y). CrT(⁻/y) mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder. PMID:21249153

  15. Ceruloplasmin/Hephaestin Knockout Mice Model Morphologic and Molecular Features of AMD

    PubMed Central

    Hadziahmetovic, Majda; Dentchev, Tzvete; Song, Ying; Haddad, Nadine; He, Xining; Hahn, Paul; Pratico, Domenico; Wen, Rong; Harris, Z. Leah; Lambris, John D.; Beard, John; Dunaief, Joshua L.

    2008-01-01

    Purpose Iron is an essential element in human metabolism but also is a potent generator of oxidative damage with levels that increase with age. Several studies suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). In prior studies, both iron overload and features of AMD were identified in mice deficient in the ferroxidase ceruloplasmin (Cp) and its homologue hephaestin (Heph) (double knockout, DKO). In this study, the location and timing of iron accumulation, the rate and reproducibility of retinal degeneration, and the roles of oxidative stress and complement activation were determined. Methods Morphologic analysis and histochemical iron detection by Perls' staining was performed on retina sections from DKO and control mice. Immunofluorescence and immunohistochemistry were performed with antibodies detecting activated complement factor C3, transferrin receptor, L-ferritin, and macrophages. Tissue iron levels were measured by atomic absorption spectrophotometry. Isoprostane F2α-VI, a specific marker of oxidative stress, was quantified in the tissue by gas chromatography/mass spectrometry. Results DKOs exhibited highly reproducible age-dependent iron overload, which plateaued at 6 months of age, with subsequent progressive retinal degeneration continuing to at least 12 months. The degeneration shared some features of AMD, including RPE hypertrophy and hyperplasia, photoreceptor degeneration, subretinal neovascularization, RPE lipofuscin accumulation, oxidative stress, and complement activation. Conclusions DKOs have age-dependent iron accumulation followed by retinal degeneration modeling some of the morphologic and molecular features of AMD. Therefore, these mice are a good platform on which to test therapeutic agents for AMD, such as antioxidants, iron chelators, and antiangiogenic agents. PMID:18326691

  16. Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

    PubMed Central

    Wood, Laura; Booth, Daniel G.; Vargiu, Giulia; Ohta, Shinya; deLima Alves, Flavia; Samejima, Kumiko; Fukagawa, Tatsuo; Rappsilber, Juri; Earnshaw, William C.

    2016-01-01

    Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis). PMID:26791246

  17. Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

    PubMed Central

    Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily

    2014-01-01

    FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA− strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569

  18. Cognitive abnormalities and hippocampal alterations in monoamine oxidase A and B knockout mice

    PubMed Central

    Singh, Chanpreet; Bortolato, Marco; Bali, Namrata; Godar, Sean C.; Scott, Anna L.; Chen, Kevin; Thompson, Richard F.; Shih, Jean C.

    2013-01-01

    The monoamine oxidase isoenzymes (MAOs) A and B play important roles in the homeostasis of monoaminergic neurotransmitters. The combined deficiency of MAO A and B results in significantly elevated levels of serotonin (5-hydroxytryptamine), norepinephrine, dopamine, and β-phenylethylamine; in humans and mice, these neurochemical changes are accompanied by neurodevelopmental perturbations as well as autistic-like responses. Ample evidence indicates that normal levels of monoamines in the hippocampus, amygdala, frontal cortex, and cerebellum are required for the integrity of learning and memory. Thus, in the present study, the cognitive status of MAO A/B knockout (KO) mice was examined with a wide array of behavioral tests. In comparison with male wild-type littermates, MAO A/B KO mice exhibited abnormally high and overgeneralized fear conditioning and enhanced eye-blink conditioning. These alterations were accompanied by significant increases in hippocampal long-term potentiation and alterations in the relative expression of NMDA glutamate receptor subunits. Our data suggest that chronic elevations of monoamines, because of the absence of MAO A and MAO B, cause functional alterations that are accompanied with changes in the cellular mechanisms underlying learning and memory. The characteristics exhibited by MAO A/B KO mice highlight the potential of these animals as a useful tool to provide further insight into the molecular bases of disorders associated with abnormal monoaminergic profiles. PMID:23858446

  19. [Metabolic shift of Corynebacterium acetoacidophilum-deltaldh under oxygen deprivation conditions].

    PubMed

    Yang, Qian; Zheng, Pu; Yu, Fang; Liu, Wei; Sun, Zhihao

    2014-03-01

    Lactate and succinate were produced by Corynebacterium acetoacidophilum from glucose under oxygen deprivation conditions. To construct knockout mutant, lactate dehydrogenase gene (ldh) of C. acetoacidophilum was deleted by double-crossover chromosome replacement with sacB gene. Comparing with the wild strain ATCC13870, ldhA-deficent mutant produced no lactate with glucose consumption rate decreased by 29.3%, while succinate and acetate concentrations were increased by 45.6% and 182%, respectively. Moreover, the NADH/NAD+ rate was less than 1 (about 0.7), and the activities of phosphoenolpyruvate carboxylase and acetate kinase of the ldhA-deficent mutant were enhanced by 84% and 12 times, respectively. Our studies show that succinicate and acetate production pathways are strengthened by blocking lactate synthesis. It also suggests that improving NADH supply and eliminating acetate generation are alternative strategies to get high succinate-producer. PMID:25007579

  20. Reductive Dechlorination of Chlorinated Ethenes Under Oxidation-Reduction Conditions and Potentiometric Surfaces in Two Trichloroethene-Contaminated Zones at the Double Eagle and Fourth Street Superfund Sites in Oklahoma City, Oklahoma

    USGS Publications Warehouse

    Braun, Christopher L.

    2004-01-01

    The Double Eagle Refining Superfund site and the Fourth Street Abandoned Refinery Superfund site are in northeast Oklahoma City, Oklahoma, adjacent to one another. The Double Eagle facility became a Superfund site on the basis of contamination from lead and volatile organic compounds; the Fourth Street facility on the basis of volatile organic compounds, pesticides, and acid-base neutral compounds. The study documented in this report was done to investigate whether reductive dechlorination of chlorinated ethenes under oxidation-reduction conditions is occurring in two zones of the Garber-Wellington aquifer (shallow zone 30?60 to 75 feet below land surface, deep zone 75 to 160 feet below land surface) at the sites; and to construct potentiometric surfaces of the two water-yielding zones to determine the directions of ground-water flow at the sites. The presence in some wells of intermediate products of reductive dechlorination, dichloroethene and vinyl chloride, is an indication that reductive dechlorination of trichloroethene is occurring. Dissolved oxygen concentrations (less than 0.5 milligram per liter) indicate that consumption of dissolved oxygen likely had occurred in the oxygen-reducing microbial process associated with reductive dechlorination. Concentrations of nitrate and nitrite nitrogen (generally less than 2.0 and 0.06 milligrams per liter, respectively) indicate that nitrate reduction probably is not a key process in either aquifer zone. Concentrations of ferrous iron greater than 1.00 milligram per liter in the majority of wells sampled indicate that iron reduction is probable. Concentrations of sulfide less than 0.05 milligram per liter in all wells indicate that sulfate reduction probably is not a key process in either zone. The presence of methane in ground water is an indication of strongly reducing conditions that facilitate reductive dechlorination. Methane was detected in all but one well. In the shallow zone in the eastern part of the study

  1. Adaptation of the myoglobin knockout mouse to hypoxic stress.

    PubMed

    Schlieper, Georg; Kim, Jie-Hoon; Molojavyi, Andrei; Jacoby, Christoph; Laussmann, Tim; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2004-04-01

    Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice we e exposed to hypoxia (10% O(2)) fo 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1alpha remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate du ing hypoxic st ess but a e surprisingly well adapted. Changes in ene gy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice. PMID:14656764

  2. Drop tests of the Three Mile Island knockout canister

    SciTech Connect

    Box, W.D.; Aaron, W.S.; Shappert, L.B.; Childress, P.C.; Quinn, G.J.; Smith, J.V.

    1986-09-01

    A type of Three Mile Island Unit 2 (TMI-2) defueling canister, called a ''knockout'' canister, was subjected to a series of drop tests at the Oak Ridge National Laboratory's Drop Test Facility. These tests were designed to confirm the structural integrity of internal fixed neutron poisons in support of a request for NRC licensing of this type of canister for the shipment of TMI-2 reactor fuel debris to the Idaho National Engineering Laboratory (INEL) for the Core Examination R and D Program. Work conducted at the Oak Ridge National Laboratory included (1) precise physical measurements of the internal poison rod configuration before assembly, (2) canister assembly and welding, (3) nondestructive examination (an initial hydrostatic pressure test and an x-ray profile of the internals before and after each drop test), (4) addition of a simulated fuel load, (5) instrumentation of the canister for each drop test, (6) fabrication of a cask simulation vessel with a developed and tested foam impact limiter, (7) use of refrigeration facilities to cool the canister to well below freezing prior to three of the drops, (8) recording the drop test with still, high-speed, and normal-speed photography, (9) recording the accelerometer measurements during impact, (10) disassembly and post-test examination with precise physical measurements, and (11) preparation of the final report.

  3. Characteristics of Skeletal Muscle Fibers of SOD1 Knockout Mice.

    PubMed

    Nagahisa, Hiroshi; Okabe, Kazuma; Iuchi, Yoshihito; Fujii, Junichi; Miyata, Hirofumi

    2016-01-01

    Cu/Zn superoxide dismutase (SOD1) knockout (KO) mice are known as an aging model in some aspects, but the damage and regeneration process of each fiber type have not been sufficiently studied. In this study, we investigated the damage and satellite cell state of the gastrocnemius muscle in SOD1 KO mice (6 months old) using immunohistochemical staining and real-time RT-PCR. The proportion of central nuclei-containing Type IIx/b fibers in the deep and superficial portions of the gastrocnemius muscle was significantly higher in SOD1 KO than control mice. The number of satellite cells per muscle fiber decreased in all muscle fiber types in the deep portion of the gastrocnemius muscle in SOD1 KO mice. In addition, the mRNA expression levels of Pax7 and myogenin, which are expressed in satellite cells in the activation, proliferation, and differentiation states, significantly increased in the gastrocnemius muscle of SOD1 KO mice. Furthermore, mRNA of myosin heavy chain-embryonic, which is expressed in the early phase of muscle regeneration, significantly increased in SOD1 KO mice. It was suggested that muscle is damaged by reactive oxygen species produced in the mitochondrial intermembrane space in Type IIxb fibers, accelerating the proliferation and differentiation of satellite cells through growth factors in SOD1 KO mice. PMID:26798428

  4. Knockout of Foxp2 disrupts vocal development in mice

    PubMed Central

    Castellucci, Gregg A.; McGinley, Matthew J.; McCormick, David A.

    2016-01-01

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/−) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/− mice. In comparison to their WT littermates, Foxp2+/− mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/− song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene’s role in general vocal motor control. PMID:26980647

  5. Improved microarray methods for profiling the yeast knockout strain collection

    PubMed Central

    Yuan, Daniel S.; Pan, Xuewen; Ooi, Siew Loon; Peyser, Brian D.; Spencer, Forrest A.; Irizarry, Rafael A.; Boeke, Jef D.

    2005-01-01

    A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens. PMID:15994458

  6. Social dominance in male vasopressin 1b receptor knockout mice.

    PubMed

    Caldwell, Heather K; Dike, Obianuju E; Stevenson, Erica L; Storck, Kathryn; Young, W Scott

    2010-07-01

    We have previously reported that mice with a targeted disruption of their vasopressin 1b receptor gene, Avpr1b, have mild impairments in social recognition and reduced aggression. The reductions in aggression are limited to social forms of aggression, i.e., maternal and inter-male aggression, while predatory aggression remains unaffected. To further clarify the role of the Avpr1b in the regulation of social behavior we first examined anxiety-like and depression-like behaviors in Avpr1b knockout (Avpr1b -/-) mice. We then went on to test the ability of Avpr1b -/- mice to form dominance hierarchies. No major differences were found between Avpr1b -/- and wildtype mice in anxiety-like behaviors, as measured using an elevated plus maze and an open field test, or depression-like behaviors, as measured using a forced swim test. In the social dominance study we found that Avpr1b -/- mice are able to form dominance hierarchies, though in early hierarchy formation dominant Avpr1b -/- mice display significantly more mounting behavior on Day 1 of testing compared to wildtype controls. Further, non-socially dominant Avpr1b -/- mice spend less time engaged in attack behavior than wildtype controls. These findings suggest that while Avpr1b -/- mice may be able to form dominance hierarchies they appear to employ alternate strategies. PMID:20298692

  7. Bone growth and turnover in progesterone receptor knockout mice.

    SciTech Connect

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O'Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  8. Immunosympathectomy as the first phenotypic knockout with antibodies

    PubMed Central

    Cattaneo, Antonino

    2013-01-01

    In a PNAS Classic Article published in 1960, Rita Levi-Montalcini offered formal and conclusive proof that endogenous NGF was responsible for the survival of sympathetic neurons in vivo. Thus ended an experimental tour de force lasting a decade, starting with the demonstration that a humoral factor, produced from a tumor transplanted in a chicken embryo, was responsible for stimulating outgrowth of nerve fibers from sympathetic and sensory neurons. From a more general methodological point of view, this work provided a breakthrough in the quest to achieve targeted loss of function and experimentally validate the function of biological molecules. Finally, this work provided an example of the ablation of a specific neuronal subpopulation in an otherwise intact nervous system, an immunological knife of unsurpassed effectiveness and precision. The novelty and the importance of the PNAS Classic Article is discussed here, collocating it within the context of the particular moment of the NGF discovery saga, of Rita Levi-Montalcini's scientific and academic career, and of the general scientific context of those years. This seminal work, involving the use of antibodies for phenotypic knockout in vivo, planted seeds that were to bear new fruit many years later with the advent of monoclonal antibodies and recombinant antibody technologies. PMID:23515328

  9. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  10. Bone Growth and Turnover in Progesterone Receptor Knockout Mice

    PubMed Central

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jamie C.; Waters, Katrina M.; Lydon, John P.; O’Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-01-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and microcomputed tomography analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 wk of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain, and tibia longitudinal bone growth were normal in PRKO mice. In contrast, total, cancellous, and cortical bone mass were increased in the humerus of 12-wk-old PRKO mice, whereas cortical and cancellous bone mass in the tibia was normal. At 26 wk of age, cancellous bone area in the proximal tibia metaphysis of PRKO mice was 153% greater than age matched wild-type mice. The improved cancellous bone balance in 6-month-old PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice is not essential for bone growth and turnover. However, at some skeletal sites, PR signaling attenuates the accumulation of cortical and cancellous bone mass during adolescence. PMID:18276762

  11. Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

    PubMed Central

    Sun, Xingshen; Olivier, Alicia K.; Yi, Yaling; Pope, Christopher E.; Hayden, Hillary S.; Liang, Bo; Sui, Hongshu; Zhou, Weihong; Hager, Kyle R.; Zhang, Yulong; Liu, Xiaoming; Yan, Ziying; Fisher, John T.; Keiser, Nicholas W.; Song, Yi; Tyler, Scott R.; Goeken, J. Adam; Kinyon, Joann M.; Radey, Matthew C.; Fligg, Danielle; Wang, Xiaoyan; Xie, Weiliang; Lynch, Thomas J.; Kaminsky, Paul M.; Brittnacher, Mitchell J.; Miller, Samuel I.; Parekh, Kalpaj; Meyerholz, David K.; Hoffman, Lucas R.; Frana, Timothy; Stewart, Zoe A.; Engelhardt, John F.

    2015-01-01

    Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients. PMID:24637292

  12. Technical knockout, a Drosophila model of mitochondrial deafness.

    PubMed Central

    Toivonen, J M; O'Dell, K M; Petit, N; Irvine, S C; Knight, G K; Lehtonen, M; Longmuir, M; Luoto, K; Touraille, S; Wang, Z; Alziari, S; Shah, Z H; Jacobs, H T

    2001-01-01

    Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity. PMID:11560901

  13. Subregion-Specific p300 Conditional Knock-Out Mice Exhibit Long-Term Memory Impairments

    ERIC Educational Resources Information Center

    Oliveira, Ana M. M.; Estevez, Marcel A.; Hawk, Joshua D.; Grimes, Shannon; Brindle, Paul K.; Abel, Ted

    2011-01-01

    Histone acetylation plays a critical role during long-term memory formation. Several studies have demonstrated that the histone acetyltransferase (HAT) CBP is required during long-term memory formation, but the involvement of other HAT proteins has not been extensively investigated. The HATs CBP and p300 have at least 400 described interacting…

  14. Conditional knockout mice demonstrate function of Klf5 as a myeloid transcription factor.

    PubMed

    Shahrin, Nur Hezrin; Diakiw, Sonya; Dent, Lindsay A; Brown, Anna L; D'Andrea, Richard J

    2016-07-01

    Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP. Using an in vivo hematopoietic-specific gene ablation model, we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) were all significantly reduced in Klf5(Δ/Δ) mice, and knockdown of KLF5 in human CD34(+) cells suppressed colony-forming potential. ST-HSCs, MPPs, and total numbers of committed progenitors were increased in the spleen of Klf5(Δ/Δ) mice, and reduced β1- and β2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis results from increased stem and progenitor mobilization. Klf5(Δ/Δ) mice show a significant reduction in the fraction of Gr1(+)Mac1(+) cells (neutrophils) in peripheral blood and bone marrow and increased frequency of eosinophils in the peripheral blood, bone marrow, and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice after GMP, promoting increased neutrophil output at the expense of eosinophil production. PMID:27207790

  15. Hair follicle defects and squamous cell carcinoma formation in Smad4 conditional knockout mouse skin.

    PubMed

    Qiao, W; Li, A G; Owens, P; Xu, X; Wang, X-J; Deng, C-X

    2006-01-12

    Smad4 is the common mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in skin development and epidermal tumorigenesis, we disrupted this gene in skin using the Cre-loxP approach. We showed that absence of Smad4 blocked hair follicle differentiation and cycling, leading to a progressive hair loss of mutant (MT) mice. MT hair follicles exhibited diminished expression of Lef1, and increased proliferative cells in the outer root sheath. Additionally, the skin of MT mice exhibited increased proliferation of basal keratinocytes and epidermal hyperplasia. Furthermore, we provide evidence that the absence of Smad4 resulted in a block of both TGFbeta and bone morphogenetic protein (BMP) signaling pathways, including p21, a well-known cyclin-dependent kinase inhibitor. Consequently, all MT mice developed spontaneous malignant skin tumors from 3 months to 13 months of age. The majority of tumors are malignant squamous cell carcinomas. A most notable finding is that tumorigenesis is accompanied by inactivation of phosphatase and tensin homolog deleted on chromosome 10 (Pten), activation of AKT, fast proliferation and nuclear accumulation of cyclin D1. These observations revealed the essential functions of Smad4-mediated signals in repressing skin tumor formation through the TGFbeta/BMP pathway, which interacts with the Pten signaling pathway. PMID:16170355

  16. Seeing Double

    NASA Astrophysics Data System (ADS)

    Pesic, Peter

    2003-10-01

    The separateness and connection of individuals is perhaps the central question of human life: What, exactly, is my individuality? To what degree is it unique? To what degree can it be shared, and how? To the many philosophical and literary speculations about these topics over time, modern science has added the curious twist of quantum theory, which requires that the elementary particles of which everything consists have no individuality at all. All aspects of chemistry depend on this lack of individuality, as do many branches of physics. From where, then, does our individuality come? In Seeing Double, Peter Pesic invites readers to explore this intriguing set of questions. He draws on literary and historical examples that open the mind (from Homer to Martin Guerre to Kafka), philosophical analyses that have helped to make our thinking and speech more precise, and scientific work that has enabled us to characterize the phenomena of nature. Though he does not try to be all-inclusive, Pesic presents a broad range of ideas, building toward a specific point of view: that the crux of modern quantum theory is its clash with our ordinary concept of individuality. This represents a departure from the usual understanding of quantum theory. Pesic argues that what is bizarre about quantum theory becomes more intelligible as we reconsider what we mean by individuality and identity in ordinary experience. In turn, quantum identity opens a new perspective on us. Peter Pesic is a Tutor and Musician-in-Residence at St. John's College, Santa Fe, New Mexico. He has a Ph.D. in physics from Stanford University.

  17. Double inflation

    SciTech Connect

    Silk, J.; Turner, M.S.

    1986-04-01

    The Zel'dovich spectrum of adiabatic density perturbations is a generic prediction of inflation. There is increasing evidence that when the spectrum is normalized by observational data on small scales, there is not enough power on large scales to account for the observed large-scale structure in the Universe. Decoupling the spectrum on large and small scales could solve this problem. As a means of decoupling the large and small scales we propose double inflation (i.e., two episodes of inflation). In this scenario the spectrum on large scales is determined by the first episode of inflation and those on small scales by a second episode of inflation. We present three models for such a scenario. By nearly saturating the large angular-scale cosmic microwave anisotropy bound, we can easily account for the observed large-scale structure. We take the perturbations on small scales to be very large, deltarho/rho approx. = 0.1 to 0.01, which results in the production of primordial black holes (PBHs), early formation of structure, reionization of the Universe, and a rich array of astrophysical events. The ..cap omega..-problem is also addressed by our scenario. Allowing the density perturbations produced by the second episode of inflation to be large also lessens the fine-tuning required in the scalar potential and makes reheating much easier. We briefly speculate on the possibility that the second episode of inflation proceeds through the nucleation of bubbles, which today manifest themselves as empty bubbles whose surfaces are covered with galaxies. 37 refs., 1 fig.

  18. Paigen Diet–Fed Apolipoprotein E Knockout Mice Develop Severe Pulmonary Hypertension in an Interleukin-1–Dependent Manner

    PubMed Central

    Lawrie, Allan; Hameed, Abdul G.; Chamberlain, Janet; Arnold, Nadine; Kennerley, Aneurin; Hopkinson, Kay; Pickworth, Josephine; Kiely, David G.; Crossman, David C.; Francis, Sheila E.

    2011-01-01

    Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have described PAH in fat-fed apolipoprotein E knockout (ApoE−/−) mice. We have reported that signaling in interleukin-1–receptor–knockout (IL-1R1−/−) mice leads to a reduction in diet-induced systemic atherosclerosis. We subsequently hypothesized that double-null (ApoE−/−/IL-1R1−/−) mice would show a reduced PAH phenotype compared with that of ApoE−/− mice. Male IL-1R1−/−, ApoE−/−, and ApoE−/−/IL-1R1−/− mice were fed regular chow or a high-fat diet (Paigen diet) for 8 weeks before phenotyping for PAH. No abnormal phenotype was observed in the IL-1R1−/− mice. Fat-fed ApoE−/− mice developed significantly increased right ventricular systolic pressure and substantial pulmonary vascular remodeling. Surprisingly, ApoE−/−/IL-1R1−/− mice showed an even more severe PAH phenotype. Further molecular investigation revealed the expression of a putative, alternatively primed IL-1R1 transcript expressed within the lungs but not aorta of ApoE−/−/IL-1R1−/− mice. Treatment of ApoE−/− and ApoE−/−/IL-1R1−/− mice with IL-1–receptor antagonist prevented progression of the PAH phenotype in both strains. Blocking IL-1 signaling may have beneficial effects in treating PAH, and alternative IL-1–receptor signaling in the lung may be important in driving PAH pathogenesis. PMID:21835155

  19. Higher Early Monocyte and Total Lymphocyte Counts Are Associated with Better Overall Survival after Standard Total Body Irradiation, Cyclophosphamide, and Fludarabine Reduced-Intensity Conditioning Double Umbilical Cord Blood Allogeneic Stem Cell Transplantation in Adults.

    PubMed

    Le Bourgeois, Amandine; Peterlin, Pierre; Guillaume, Thierry; Delaunay, Jacques; Duquesne, Alix; Le Gouill, Steven; Moreau, Philippe; Mohty, Mohamad; Campion, Loïc; Chevallier, Patrice

    2016-08-01

    This single-center retrospective study aimed to report the impact of early hematopoietic and immune recoveries after a standard total body irradiation, cyclophosphamide, and fludarabine (TCF) reduced-intensity conditioning (RIC) regimen for double umbilical cord blood (dUCB) allogeneic stem cell transplantation (allo-SCT) in adults. We analyzed 47 consecutive patients older than 17 years who engrafted after a dUCB TCF allo-SCT performed between January 2006 and April 2013 in our department. Median times for neutrophil and platelet recoveries were 17 (range, 6 to 59) and 37 days (range, 0 to 164), respectively. The 3-year overall (OS) and disease-free survivals, relapse incidence, and nonrelapse mortality were 65.7%, 57.2%, 27.1%, and 19%, respectively. In multivariate analysis, higher day +30 monocyte (≥615/mm(3); hazard ratio [HR], .04; 95% confidence interval [CI], .004 to .36; P < .01) and day +42 lymphocyte (≥395/mm(3); HR, .16; 95% CI, .03 to .78; P = .02) counts were independently associated with better OS. These results suggest that early higher hematopoietic and immune recovery is predictive of survival after dUCB TCF RIC allo-SCT in adults. Factors other than granulocyte colony-stimulating factor, which was used in all cases, favoring expansion of monocytes or lymphocytes, should be tested in the future as part of the UCB transplantation procedure. PMID:27118570

  20. 3-D Modeling of Double-Diffusive Convection During Directional Solidification of a Non-Dilute Alloy with Application to the HgCdTe Growth Under Microgravity Conditions

    NASA Technical Reports Server (NTRS)

    Bune, Andris V.; Gillies, Donald C.; Lehoczky, Sandor L.

    1998-01-01

    A numerical calculation for a non-dilute alloy solidification was performed using the FIDAP finite element code. For low growth velocities plane front solidification occurs. The location and the shape of the interface was determined using melting temperatures from the HgCdTe liquidus curve. The low thermal conductivity of the solid HgCdTe causes thermal short circuit through the ampoule walls, resulting in curved isotherms in the vicinity of the interface. Double-diffusive convection in the melt is caused by radial temperature gradients and by material density inversion with temperature. Cooling from below and the rejection at the solid-melt interface of the heavier HgTe-rich solute each tend to reduce convection. Because of these complicating factors dimensional rather then non-dimensional modeling was performed. Estimates of convection contributions for various gravity conditions was performed parametrically. For gravity levels higher then 1 0 -7 of earth's gravity it was found that the maximum convection velocity is extremely sensitive to gravity vector orientation and can be reduced at least by factor of 50% for precise orientation of the ampoule in the microgravity environment. The predicted interface shape is in agreement with one obtained experimentally by quenching. The results of 3-D modeling are compared with previous 2-D finding. A video film featuring melt convection will be presented.

  1. Muscular pre-conditioning using light-emitting diode therapy (LEDT) for high-intensity exercise: a randomized double-blind placebo-controlled trial with a single elite runner

    PubMed Central

    Ferraresi, Cleber; Beltrame, Thomas; Fabrizzi, Fernando; do Nascimento, Eduardo Sanches Pereira; Karsten, Marlus; de Oliveira Francisco, Cristina; Borghi-Silva, Audrey; Catai, Aparecida Maria; Cardoso, Daniel Rodrigues; Ferreira, Antonio Gilberto; Hamblin, Michael R.; Bagnato, Vanderlei Salvador; Parizotto, Nivaldo Antonio

    2015-01-01

    Recently, low-level laser (light) therapy (LLLT) has been used to improve muscle performance. This study aimed to evaluate the effectiveness of near-infrared light-emitting diode therapy (LEDT) and its mechanisms of action to improve muscle performance in an elite athlete. The kinetics of oxygen uptake (VO2), blood and urine markers of muscle damage (creatine kinase – CK and alanine) and fatigue (lactate) were analyzed. Additionally, some metabolic parameters were assessed in urine using proton nuclear magnetic resonance spectroscopy (1H NMR). A LED cluster with 50 LEDs (λ = 850 nm; 50mW 15 s; 37.5 J) was applied on legs, arms and trunk muscles of a single runner athlete 5 min before a high-intense constant workload running exercise on treadmill. The athlete received either Placebo-1-LEDT; Placebo-2-LEDT; or Effective-LEDT in a randomized double-blind placebo-controlled trial with washout period of 7 d between each test. LEDT improved the speed of the muscular VO2 adaptation (~−9 s), decreased O2 deficit (~−10 L), increased the VO2 from the slow component phase (~+348 ml min−1) and increased the time limit of exercise (~+589 s). LEDT decreased blood and urine markers of muscle damage and fatigue (CK, alanine and lactate levels). The results suggest that a muscular pre-conditioning regimen using LEDT before intense exercises could modulate metabolic and renal function to achieve better performance. PMID:25585514

  2. Loss of Urinary Macromolecules in Mice Causes Interstitial and Intratubular Renal Calcification Dependent on the Underlying Conditions

    NASA Astrophysics Data System (ADS)

    Wu, Xue-Ru; Lieske, John C.; Evan, Andrew P.; Sommer, Andre J.; Liaw, Lucy; Mo, Lan

    2008-09-01

    Urinary protein macromolecules have long been thought to play a role in influencing the various phases of urolithiasis including nucleation, growth, aggregation of mineral crystals and their subsequent adhesion to the renal epithelial cells. However, compelling evidence regarding their precise role was lacking, due partly to the fact that most prior studies were done in vitro and results were highly variable depending on the experimental conditions. The advent of genetic engineering technology has made it possible to study urinary protein macromolecules within an in vivo biological system. Indeed, recent studies have begun to shed light on the net effects of loss of one or more macromolecules on the earliest steps of urolithiasis. This paper focuses on the in vivo consequences of inactivating Tamm-Horsfall protein and/or osteopontin, two major urinary glycoproteins, using the knockout approach. The renal phenotypes of both single and double knockout mice under spontaneous or hyperoxaluric conditions will be described. The functional significance of the urinary macromolecules as critical defense factors against renal calcification will also be discussed.

  3. Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders

    PubMed Central

    Lim, Elaine T.; Raychaudhuri, Soumya; Sanders, Stephan J.; Stevens, Christine; Sabo, Aniko; MacArthur, Daniel G.; Neale, Benjamin M.; Kirby, Andrew; Ruderfer, Douglas M.; Fromer, Menachem; Lek, Monkol; Liu, Li; Flannick, Jason; Ripke, Stephan; Nagaswamy, Uma; Muzny, Donna; Reid, Jeffrey G.; Hawes, Alicia; Newsham, Irene; Wu, Yuanqing; Lewis, Lora; Dinh, Huyen; Gross, Shannon; Wang, Li-San; Lin, Chiao-Feng; Valladares, Otto; Gabriel, Stacey B.; dePristo, Mark; Altshuler, David M.; Purcell, Shaun M.; State, Matthew W.; Boerwinkle, Eric; Buxbaum, Joseph D.; Cook, Edwin H.; Gibbs, Richard A.; Schellenberg, Gerard D.; Sutcliffe, James S.; Devlin, Bernie; Roeder, Kathryn; Daly, Mark J.

    2013-01-01

    SUMMARY To characterize the role of rare complete human knockouts in autism spectrum disorders (ASD), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a two-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudo-autosomal regions on the X-chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together these results provide compelling evidence that rare autosomal and X-chromosome complete gene knockouts are important inherited risk factors for ASD. PMID:23352160

  4. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  5. Guwiyang Wurra--'Fire Mouse': a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies.

    PubMed

    Middleton, Ryan J; Liu, Guo-Jun; Banati, Richard B

    2015-08-01

    The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands. PMID:26551692

  6. Apolipoprotein E knockout as the basis for mouse models of dyslipidemia-induced neuropathy.

    PubMed

    Hinder, Lucy M; Vincent, Andrea M; Hayes, John M; McLean, Lisa L; Feldman, Eva L

    2013-01-01

    Dyslipidemia has been identified as an important pathogenic risk factor for diabetic neuropathy, but current animal models do not adequately reproduce the lipid profile observed in human diabetics (increased triglycerides with an elevated LDL-cholesterol and reduced HDL-cholesterol). High fat feeding of mice produces hyperlipidemia, but mice are resistant to increases in the LDL to HDL ratio, reducing the potential for peripheral lipid deposits to impact neuropathy, as is postulated to occur in human subjects. Genetic manipulations provide an alternative approach to reproducing a neuropathic plasma lipid profile. Based on findings from the atherosclerosis literature, we began with knockout of ApoE. Since knockout of ApoE alone only partially mimics the human diabetic lipid profile, we examined the impact of its combination with a well-characterized model of type 2 diabetes exhibiting neuropathy, the db/db mouse. We added further gene manipulations to increase hyperlipidemia by using mice with both ApoE and ApoB48 knockout on the ob/+ (leptin mutation) mice. In all of these models, we found that either the db/db or ob/ob genotypes had increased body weight, hyperlipidemia, hyperglycemia, and evidence of neuropathy compared with the control groups (db/+ or ob/+, respectively). We found that ApoE knockout combined with leptin receptor knockout produced a lipid profile most closely modeling human dyslipidemia that promotes neuropathy. ApoE knockout combined with additional ApoB48 and leptin knockout produced similar changes of smaller magnitude, but, notably, an increase in HDL-cholesterol. Our data suggest that the overall effects of ApoE knockout, either directly upon nerve structure and function or indirectly on lipid metabolism, are insufficient to significantly alter the course of diabetic neuropathy. Although these models ultimately do not deliver optimal lipid profiles for translational diabetic neuropathy research, they do present glycemic and lipid profile

  7. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii. PMID

  8. 2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions1

    PubMed Central

    Awad, Jasmin; Stotz, Henrik U.; Fekete, Agnes; Krischke, Markus; Engert, Cornelia; Havaux, Michel; Berger, Susanne; Mueller, Martin J.

    2015-01-01

    Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX. PMID:25667319

  9. Enhanced colonic tumorigenesis in alkaline sphingomyelinase (NPP7) knockout mice.

    PubMed

    Chen, Ying; Zhang, Ping; Xu, Shu-Chang; Yang, Liping; Voss, Ulrikke; Ekblad, Eva; Wu, Yunjin; Min, Yalan; Hertervig, Erik; Nilsson, Åke; Duan, Rui-Dong

    2015-01-01

    Intestinal alkaline sphingomyelinase (alk-SMase) generates ceramide and inactivates platelet-activating factor (PAF) and was previously suggested to have anticancer properties. The direct evidence is still lacking. We studied colonic tumorigenesis in alk-SMase knockout (KO) mice. Formation of aberrant crypt foci (ACF) was examined after azoxymethane (AOM) injection. Tumor was induced by AOM alone, a conventional AOM/dextran sulfate sodium (DSS) treatment, and an enhanced AOM/DSS method. β-Catenin was determined by immunohistochemistry, PAF levels by ELISA, and sphingomyelin metabolites by mass spectrometry. Without treatment, spontaneous tumorigenesis was not identified but the intestinal mucosa appeared thicker in KO than in wild-type (WT) littermates. AOM alone induced more ACF in KO mice but no tumors 28 weeks after injection. However, combination of AOM/DSS treatments induced colonic tumors and the incidence was significantly higher in KO than in WT mice. By the enhanced AOM/DSS method, tumor number per mouse increased 4.5 times and tumor size 1.8 times in KO compared with WT mice. Although all tumors were adenomas in WT mice, 32% were adenocarcinomas in KO mice. Compared with WT mice, cytosol expression of β-catenin was significantly increased and nuclear translocation in tumors was more pronounced in KO mice. Lipid analysis showed decreased ceramide in small intestine and increased sphingosine-1-phosphate (S1P) in both small intestine and colon in nontreated KO mice. PAF levels in feces were significantly higher in the KO mice after AOM/DSS treatment. In conclusion, lack of alk-SMase markedly increases AOM/DSS-induced colonic tumorigenesis associated with decreased ceramide and increased S1P and PAF levels. PMID:25381265

  10. Feeding-elicited cataplexy in orexin knockout mice

    PubMed Central

    Clark, Erika L.; Baumann, Christian R.; Cano, Georgina; Scammell, Thomas E.; Mochizuki, Takatoshi

    2009-01-01

    Mice lacking orexin/hypocretin signaling have sudden episodes of atonia and paralysis during active wakefulness. These events strongly resemble cataplexy, episodes of sudden muscle weakness triggered by strong positive emotions in people with narcolepsy, but it remains unknown whether murine cataplexy is triggered by positive emotions. To determine whether positive emotions elicit murine cataplexy, we placed orexin knockout (KO) mice on a scheduled feeding protocol with regular or highly palatable food. Baseline sleep/wake behavior was recorded with ad lib regular chow. Mice were then placed on a scheduled feeding protocol in which they received 60% of their normal amount of chow 3 hr after dark onset for the next 10 days. Wild-type and KO mice rapidly entrained to scheduled feeding with regular chow, with more wake and locomotor activity prior to the feeding time. On day 10 of scheduled feeding, orexin KO mice had slightly more cataplexy during the food-anticipation period and more cataplexy in the second half of the dark period, when they may have been foraging for residual food. To test whether more palatable food increases cataplexy, mice were then switched to scheduled feeding with an isocaloric amount of Froot Loops, a food often used as a reward in behavioral studies. With this highly palatable food, orexin KO mice had much more cataplexy during the food-anticipation period and throughout the dark period. The increase in cataplexy with scheduled feeding, especially with highly palatable food, suggests that positive emotions may trigger cataplexy in mice, just as in people with narcolepsy. Establishing this connection helps validate orexin KO mice as an excellent model of human narcolepsy and provides an opportunity to better understand the mechanisms that trigger cataplexy. PMID:19362119

  11. Autism-related behavioral abnormalities in synapsin knockout mice

    PubMed Central

    Greco, Barbara; Managò, Francesca; Tucci, Valter; Kao, Hung-Teh; Valtorta, Flavia; Benfenati, Fabio

    2013-01-01

    Several synaptic genes predisposing to autism-spectrum disorder (ASD) have been identified. Nonsense and missense mutations in the SYN1 gene encoding for Synapsin I have been identified in families segregating for idiopathic epilepsy and ASD and genetic mapping analyses have identified variations in the SYN2 gene as significantly contributing to epilepsy predisposition. Synapsins (Syn I/II/III) are a multigene family of synaptic vesicle-associated phosphoproteins playing multiple roles in synaptic development, transmission and plasticity. Lack of SynI and/or SynII triggers a strong epileptic phenotype in mice associated with mild cognitive impairments that are also present in the non-epileptic SynIII−/− mice. SynII−/− and SynIII−/− mice also display schizophrenia-like traits, suggesting that Syns could be involved in the regulation of social behavior. Here, we studied social interaction and novelty, social recognition and social dominance, social transmission of food preference and social memory in groups of male SynI−/−, SynII−/− and SynIII−/− mice before and after the appearance of the epileptic phenotype and compared their performances with control mice. We found that deletion of Syn isoforms widely impairs social behaviors and repetitive behaviors, resulting in ASD-related phenotypes. SynI or SynIII deletion altered social behavior, whereas SynII deletion extensively impaired various aspects of social behavior and memory, altered exploration of a novel environment and increased self-grooming. Social impairments of SynI−/− and SynII−/− mice were evident also before the onset of seizures. The results demonstrate an involvement of Syns in generation of the behavioral traits of ASD and identify Syn knockout mice as a useful experimental model of ASD and epilepsy. PMID:23280234

  12. Spontaneous sleep and homeostatic sleep regulation in ghrelin knockout mice.

    PubMed

    Szentirmai, Eva; Kapás, Levente; Sun, Yuxiang; Smith, Roy G; Krueger, James M

    2007-07-01

    Ghrelin is well known for its feeding and growth hormone-releasing actions. It may also be involved in sleep regulation; intracerebroventricular administration and hypothalamic microinjections of ghrelin stimulate wakefulness in rats. Hypothalamic ghrelin, together with neuropeptide Y and orexin form a food intake-regulatory circuit. We hypothesized that this circuit also promotes arousal. To further investigate the role of ghrelin in the regulation of sleep-wakefulness, we characterized spontaneous and homeostatic sleep regulation in ghrelin knockout (KO) and wild-type (WT) mice. Both groups of mice exhibited similar diurnal rhythms with more sleep and less wakefulness during the light period. In ghrelin KO mice, spontaneous wakefulness and rapid-eye-movement sleep (REMS) were slightly elevated, and non-rapid-eye-movement sleep (NREMS) was reduced. KO mice had more fragmented NREMS than WT mice, as indicated by the shorter and greater number of NREMS episodes. Six hours of sleep deprivation induced rebound increases in NREMS and REMS and biphasic changes in electroencephalographic slow-wave activity (EEG SWA) in both genotypes. Ghrelin KO mice recovered from NREMS and REMS loss faster, and the delayed reduction in EEG SWA, occurring after sleep loss-enhanced increases in EEG SWA, was shorter-lasting compared with WT mice. These findings suggest that the basic sleep-wake regulatory mechanisms in ghrelin KO mice are not impaired and they are able to mount adequate rebound sleep in response to a homeostatic challenge. It is possible that redundancy in the arousal systems of the brain or activation of compensatory mechanisms during development allow for normal sleep-wake regulation in ghrelin KO mice. PMID:17409264

  13. Targeting cancer using KAT inhibitors to mimic lethal knockouts

    PubMed Central

    Brown, James A.L.; Bourke, Emer; Eriksson, Leif A.; Kerin, Michael J.

    2016-01-01

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  14. Diet-Induced Obesity in the Selenocysteine Lyase Knockout Mouse

    PubMed Central

    Gilman, Christy L.; Hashimoto, Ann C.; Ogawa-Wong, Ashley N.; Berry, Marla J.

    2015-01-01

    Abstract Aims: Selenocysteine lyase (Scly) mediates selenocysteine decomposition. It was previously demonstrated that, upon adequate caloric intake (12% kcal fat) and selenium deficiency, disruption of Scly in mice leads to development of metabolic syndrome. In this study, we investigate the effect of a high-fat (45% kcal) selenium-adequate diet in Scly knockout (KO) mice on development of metabolic syndrome. Involvement of selenoproteins in energy metabolism after Scly disruption was also examined in vitro in the murine hepatoma cell line, Hepa1-6, following palmitate treatment. Results: Scly KO mice were more susceptible to diet-induced obesity than their wild-type counterparts after feeding a high-fat selenium-adequate diet. Scly KO mice had aggravated hyperinsulinemia, hypercholesterolemia, glucose, and insulin intolerance, but unchanged inflammatory cytokines and expression of most selenoproteins, except increased serum selenoprotein P (Sepp1). Scly KO mice also exhibited enhanced hepatic levels of pyruvate and enzymes involved in the regulation of pyruvate cycling, such as pyruvate carboxylase (Pcx) and pyruvate dehydrogenase (Pdh). However, in vitro silencing of Scly in Hepa1-6 cells led to diminished Sepp1 expression, and concomitant palmitate treatment decreased Pdh expression. Innovation: The role of selenium in lipid metabolism is recognized, but specific selenium-dependent mechanisms leading to obesity are unclear. This study uncovers that Scly has a remarkable effect on obesity and metabolic syndrome development triggered by high-fat exposure, independent of the expression of most selenoproteins. Conclusion: Diet-induced obesity in Scly KO mice is aggravated, with effects on pyruvate levels and consequent activation of energy metabolism independent of selenoprotein levels. Antioxid. Redox Signal. 23, 761–774. PMID:26192035

  15. Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice

    PubMed Central

    Korsten, Hanneke; Ziel-van der Made, Angelique C. J.; van Weerden, Wytske M.; van der Kwast, Theo; Trapman, Jan; Van Duijn, Petra W.

    2016-01-01

    Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m) developed at older age (>10m) into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC), adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK), and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7–8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1) and tumor class 2 (TC2). TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma / intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor model

  16. Targeting cancer using KAT inhibitors to mimic lethal knockouts.

    PubMed

    Brown, James A L; Bourke, Emer; Eriksson, Leif A; Kerin, Michael J

    2016-08-15

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  17. Involvement of respiratory processes in the transient knockout of net CO2 uptake in Mimosa pudica upon heat stimulation.

    PubMed

    Lautner, Silke; Stummer, Michaela; Matyssek, Rainer; Fromm, Jörg; Grams, Thorsten E E

    2014-01-01

    Leaf photosynthesis of the sensitive plant Mimosa pudica displays a transient knockout in response to electrical signals induced by heat stimulation. This study aims at clarifying the underlying mechanisms, in particular, the involvement of respiration. To this end, leaf gas exchange and light reactions of photosynthesis were assessed under atmospheric conditions largely eliminating photorespiration by either elevated atmospheric CO2 or lowered O2 concentration (i.e. 2000 μmol mol(-1) or 1%, respectively). In addition, leaf gas exchange was studied in the absence of light. Under darkness, heat stimulation caused a transient increase of respiratory CO2 release simultaneously with stomatal opening, hence reflecting direct involvement of respiratory stimulation in the drop of the net CO2 uptake rate. However, persistence of the transient decline in net CO2 uptake rate under illumination and elevated CO2 or 1% O2 makes it unlikely that photorespiration is the metabolic origin of the respiratory CO2 release. In conclusion, the transient knockout of net CO2 uptake is at least partially attributed to an increased CO2 release through mitochondrial respiration as stimulated by electrical signals. Putative CO2 limitation of Rubisco due to decreased activity of carbonic anhydrase was ruled out as the photosynthesis effect was not prevented by elevated CO2 . PMID:23763645

  18. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    PubMed

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds. PMID:26428915

  19. AMPKα1 knockout enhances nociceptive behaviors and spinal glutamatergic synaptic activities via production of reactive oxygen species in the spinal dorsal horn.

    PubMed

    Maixner, Dylan W; Yan, Xisheng; Hooks, Shelley B; Weng, Han-Rong

    2016-06-21

    Emerging studies have shown that pharmacological activation of adenosine monophosphate-activated protein kinase (AMPK) produces potent analgesic effects in different animal pain models. Currently, the spinal molecular and synaptic mechanism by which AMPK regulates the pain signaling system remains unclear. To address this issue, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice. We demonstrated that AMPKα1 is imperative for maintaining normal nociception, and mice deficient for AMPKα1 exhibit mechanical allodynia. This is concomitantly associated with increased glutamatergic synaptic activities in neurons located in the superficial spinal dorsal horn, which results from the increased glutamate release from presynaptic terminals and function of ligand-gated glutamate receptors at the postsynaptic neurons. Additionally, AMPKα1 knockout mice have increased activities of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinases (p38), as well as elevated levels of interleukin-1β (IL-1β), reactive oxygen species (ROS), and heme oxygenase 1 (HO-1) in the spinal dorsal horn. Systemic administration of a non-specific ROS scavenger (phenyl-N-tert-butylnitrone, PBN) or a HO-1 activator (Cobalt protoporphyrin IX, CoPP) attenuated allodynia in AMPKα1 knockout mice. Bath-perfusion of the ROS scavenger or HO-1 activator effectively attenuated the increased ROS levels and glutamatergic synaptic activities in the spinal dorsal horn. Our findings suggest that ROS are the key down-stream signaling molecules mediating the behavioral hypersensitivity in AMPKα1 knockout mice. Thus, targeting AMPKα1 may represent an effective approach for the treatment of pathological pain conditions associated with neuroinflammation at the spinal dorsal horn. PMID:27058143

  20. GABAA-receptor modification in taurine transporter knockout mice causes striatal disinhibition

    PubMed Central

    Sergeeva, O A; Fleischer, W; Chepkova, A N; Warskulat, U; Häussinger, D; Siebler, M; Haas, H L

    2007-01-01

    The Striatum is involved in the regulation of movements and motor skills. We have shown previously, that the osmolyte and neuromodulator taurine plays a role in striatal plasticity. We demonstrate now that hereditary taurine deficiency in taurine-transporter knock-out (TAUT KO) mice results in disinhibition of striatal network activity, which can be corrected by taurine supplementation. Modification of GABAA but not glycine receptors (taurine is a ligand for both receptor types) underlies this disinhibition. Whole-cell recordings from acutely isolated as well as cultured striatal neurons revealed a decreased agonist sensitivity of the GABAA receptor in TAUT KO neurons in the absence of changes in the maximal GABA-evoked current amplitude. The striatal GABA level in TAUT KO mice was unchanged. The amplitude enhancement of spontaneous IPSCs by zolpidem was stronger in TAUT KO than in wild-type (WT) animals. Tonic inhibition was absent in striatal neurons under control conditions but was detected after incubation with the GABA-transaminase inhibitor vigabatrin: bicuculline induced a larger shift of baseline current in WT as compared to TAUT KO neurons. Lack of taurine leads to reduced sensitivity of synaptic and extrasynaptic GABAA receptors and consequently to disinhibition. These findings help in understanding neuropathologies accompanied by the loss of endogenous taurine, for instance in hepatic encephalopathy. PMID:17962336

  1. Dopamine beta-hydroxylase knockout mice have alterations in dopamine signaling and are hypersensitive to cocaine.

    PubMed

    Schank, Jesse R; Ventura, Rossella; Puglisi-Allegra, Stefano; Alcaro, Antonio; Cole, Charlene D; Liles, L Cameron; Seeman, Philip; Weinshenker, David

    2006-10-01

    Multiple lines of evidence demonstrate that the noradrenergic system provides both direct and indirect excitatory drive onto midbrain dopamine (DA) neurons. We used DA beta-hydroxylase (DBH) knockout (Dbh-/-) mice that lack norepinephrine (NE) to determine the consequences of chronic NE deficiency on midbrain DA neuron function in vivo. Basal extracellular DA levels were significantly attenuated in the nucleus accumbens (NAc) and caudate putamen (CP), but not prefrontal cortex (PFC), of Dbh-/- mice, while amphetamine-induced DA release was absent in the NAc and attenuated in the CP and PFC. The decrease in dopaminergic tone was associated with a profound increase in the density of high-affinity state D1 and D2 DA receptors in the NAc and CP, while DA receptors in the PFC were relatively unaffected. As a behavioral consequence of these neurochemical changes, Dbh-/- mice were hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, as measured by locomotor activity and conditioned place preference. Antagonists of DA, but not 5-HT, receptors attenuated the locomotor hypersensitivity to cocaine in Dbh-/- mice. As DBH activity in humans is genetically controlled and the DBH inhibitor disulfiram has shown promise as a pharmacotherapy for cocaine dependence, these results have implications for the influence of genetic and pharmacological DBH inhibition on DA system function and drug addiction. PMID:16395294

  2. Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression.

    PubMed

    Xie, Xie; Dubrovsky, Edward B

    2015-12-01

    RNase Z(L) is a highly conserved tRNA 3'-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase Z(L) (dRNaseZ) has a mitochondria targeting signal (MTS) flanked by two methionines at the N-terminus. Alternative translation initiation yields two protein forms: the long one is mitochondrial, and the short one may localize in the nucleus or cytosol. Here, we have generated a mitochondria specific knockout of the dRNaseZ gene. In this in vivo model, cells deprived of dRNaseZ activity display impaired mitochondrial polycistronic transcript processing, increased reactive oxygen species (ROS) and a switch to aerobic glycolysis compensating for cellular ATP. Damaged mitochondria impose a cell cycle delay at the G2 phase disrupting cell proliferation without affecting cell viability. Antioxidants attenuate genotoxic stress and rescue cell proliferation, implying a critical role for ROS. We suggest that under a low-stress condition, ROS activate tumor suppressor p53, which modulates cell cycle progression and promotes cell survival. Transcriptional profiling of p53 targets confirms upregulation of antioxidant and cycB-Cdk1 inhibitor genes without induction of apoptotic genes. This study implicates Drosophila RNase Z(L) in a novel retrograde signaling pathway initiated by the damage in mitochondria and manifested in a cell cycle delay before the mitotic entry. PMID:26553808

  3. Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice.

    PubMed

    Usuki, Takako; Takato, Tamae; Lu, Qiang; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

    2016-10-01

    Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth. PMID:27423518

  4. Impaired extinction of learned contextual fear memory in early growth response 1 knockout mice.

    PubMed

    Han, Seungrie; Hong, Soontaek; Mo, Jiwon; Lee, Dongmin; Choi, Eunju; Choi, June-seek; Sun, Woong; Lee, Hyun Woo; Kim, Hyun

    2014-01-01

    Inductive expression of early growth response 1 (Egr-1) in neurons is associated with many forms of neuronal activity. However, only a few Egr-1 target genes are known in the brain. The results of this study demonstrate that Egr-1 knockout (KO) mice display impaired contextual extinction learning and normal fear acquisition relative to wild-type (WT) control animals. Genome-wide microarray experiments revealed 368 differentially expressed genes in the hippocampus of Egr-1 WT exposed to different phases of a fear conditioning paradigm compared to gene expression profiles in the hippocampus of KO mice. Some of genes, such as serotonin receptor 2C (Htr2c), neuropeptide B (Npb), neuronal PAS domain protein 4 (Npas4), NPY receptor Y1 (Npy1r), fatty acid binding protein 7 (Fabp7), and neuropeptide Y (Npy) are known to regulate processing of fearful memories, and promoter analyses demonstrated that several of these genes contained Egr-1 binding sites. This study provides a useful list of potential Egr-1 target genes which may be regulated during fear memory processing. PMID:24552706

  5. Increased fronto-hippocampal connectivity in the Prrxl1 knockout mouse model of congenital hypoalgesia.

    PubMed

    Monteiro, Clara; Cardoso-Cruz, Helder; Matos, Mariana; Dourado, Margarida; Lima, Deolinda; Galhardo, Vasco

    2016-09-01

    Despite the large number of studies addressing how prolonged painful stimulation affects brain functioning, there are only a handful of studies aimed at uncovering if persistent conditions of reduced pain perception would also result in brain plasticity. Permanent hypoalgesia induced by neonatal injection of capsaicin or carrageenan has already been shown to affect learning and memory and to induce alterations in brain gene expression. In this study, we used the Prrxl1 model of congenital mild hypoalgesia to conduct a detailed study of the neurophysiological and behavioral consequences of reduced pain experience. Prrxl1 knockout animals are characterized by selective depletion of small diameter primary afferents and abnormal development of the superficial dorsal laminae of the spinal cord, resulting in diminished pain perception but normal tactile and motor behaviour. Behavioral testing of Prrxl1 mice revealed that these animals have reduced anxiety levels, enhanced memory performance, and improved fear extinction. Neurophysiological recordings from awake behaving Prrxl1 mice show enhanced altered fronto-hippocampal connectivity in the theta- and gamma-bands. Importantly, although inflammatory pain by Complete Freund Adjuvant injection caused a decrease in fronto-hippocampal connectivity in the wild-type animals, Prrxl1 mice maintained the baseline levels. The onset of inflammatory pain also reverted the differences in forebrain expression of stress- and monoamine-related genes in Prrxl1 mice. Altogether our results suggest that congenital hypoalgesia may have an effect on brain plasticity that is the inverse of what is usually observed in animal models of chronic pain. PMID:27168359

  6. Tubular von Hippel-Lindau Knockout Protects against Rhabdomyolysis-Induced AKI

    PubMed Central

    Fähling, Michael; Mathia, Susanne; Paliege, Alexander; Koesters, Robert; Mrowka, Ralf; Peters, Harm; Persson, Pontus Börje; Neumayer, Hans-Hellmut; Bachmann, Sebastian

    2013-01-01

    Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA–based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI. PMID:23970125

  7. Autistic-Like Traits and Cerebellar Dysfunction in Purkinje Cell PTEN Knock-Out Mice.

    PubMed

    Cupolillo, Dario; Hoxha, Eriola; Faralli, Alessio; De Luca, Annarita; Rossi, Ferdinando; Tempia, Filippo; Carulli, Daniela

    2016-05-01

    Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits. PMID:26538449

  8. Comprehensive Behavioral Analysis of Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice

    PubMed Central

    Takao, Keizo; Tanda, Koichi; Nakamura, Kenji; Kasahara, Jiro; Nakao, Kazuki; Katsuki, Motoya; Nakanishi, Kazuo; Yamasaki, Nobuyuki; Toyama, Keiko; Adachi, Minami; Umeda, Masahiro; Araki, Tsutomu; Fukunaga, Kohji; Kondo, Hisatake; Sakagami, Hiroyuki; Miyakawa, Tsuyoshi

    2010-01-01

    Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency. PMID:20209163

  9. Phospholipase D δ knock-out mutants are tolerant to severe drought stress

    PubMed Central

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  10. Benefits of a "vulnerability gene"? A study in serotonin transporter knockout mice.

    PubMed

    Kästner, Niklas; Richter, S Helene; Lesch, Klaus-Peter; Schreiber, Rebecca S; Kaiser, Sylvia; Sachser, Norbert

    2015-04-15

    Over the past years, certain "vulnerability genes" have been identified that play a key role in the development of mood and anxiety disorders. In particular, a low-expressing variant of the human serotonin transporter (5-HTT) gene has been described that renders individuals more susceptible to adverse experience and hence to the development of psychiatric diseases. However, some authors have recently argued that lower 5-HTT expression not only increases vulnerability to adverse experiences, but also enhances susceptibility to beneficial experiences, thus promoting phenotypic plasticity. The aim of the present study was to assess the effects of 5-HTT expression on susceptibility to beneficial experience in a hypothesis-driven experimental approach. Using a well-established rodent model for the human polymorphism, male heterozygous 5-HTT knockout (HET) and 5-HTT wildtype (WT) mice were either provided with the beneficial experience of cohabitation with a female (mating experience) or kept as naïve controls in single-housing conditions. Following the experimental treatment, they were tested for their anxiety-like behaviour and exploratory locomotion in three widely used behavioural tests. Interestingly, while cohabitation reduced anxiety-like behaviour and increased exploratory locomotion in the open field test in HET mice, it did not affect WT mice, pointing to a genotype-dependent susceptibility to the beneficial experience. Thus, our results might support the view of the low expressing version of the 5-HTT gene as a "plasticity" rather than a "vulnerability" variant. PMID:25629942

  11. Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression

    PubMed Central

    Xie, Xie; Dubrovsky, Edward B.

    2015-01-01

    RNase ZL is a highly conserved tRNA 3′-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase ZL (dRNaseZ) has a mitochondria targeting signal (MTS) flanked by two methionines at the N-terminus. Alternative translation initiation yields two protein forms: the long one is mitochondrial, and the short one may localize in the nucleus or cytosol. Here, we have generated a mitochondria specific knockout of the dRNaseZ gene. In this in vivo model, cells deprived of dRNaseZ activity display impaired mitochondrial polycistronic transcript processing, increased reactive oxygen species (ROS) and a switch to aerobic glycolysis compensating for cellular ATP. Damaged mitochondria impose a cell cycle delay at the G2 phase disrupting cell proliferation without affecting cell viability. Antioxidants attenuate genotoxic stress and rescue cell proliferation, implying a critical role for ROS. We suggest that under a low-stress condition, ROS activate tumor suppressor p53, which modulates cell cycle progression and promotes cell survival. Transcriptional profiling of p53 targets confirms upregulation of antioxidant and cycB-Cdk1 inhibitor genes without induction of apoptotic genes. This study implicates Drosophila RNase ZL in a novel retrograde signaling pathway initiated by the damage in mitochondria and manifested in a cell cycle delay before the mitotic entry. PMID:26553808

  12. Phospholipase D δ knock-out mutants are tolerant to severe drought stress.

    PubMed

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; Ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  13. Multiple knockout mouse models reveal lincRNAs are required for life and brain development

    PubMed Central

    Sauvageau, Martin; Goff, Loyal A; Lodato, Simona; Bonev, Boyan; Groff, Abigail F; Gerhardinger, Chiara; Sanchez-Gomez, Diana B; Hacisuleyman, Ezgi; Li, Eric; Spence, Matthew; Liapis, Stephen C; Mallard, William; Morse, Michael; Swerdel, Mavis R; D’Ecclessis, Michael F; Moore, Jennifer C; Lai, Venus; Gong, Guochun; Yancopoulos, George D; Frendewey, David; Kellis, Manolis; Hart, Ronald P; Valenzuela, David M; Arlotta, Paola; Rinn, John L

    2013-01-01

    Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr−/− neonates, whereas linc–Brn1b−/− mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001 PMID:24381249

  14. A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

    PubMed Central

    Brehm, Ralph; Zeiler, Martina; Rüttinger, Christina; Herde, Katja; Kibschull, Mark; Winterhager, Elke; Willecke, Klaus; Guillou, Florian; Lécureuil, Charlotte; Steger, Klaus; Konrad, Lutz; Biermann, Katharina; Failing, Klaus; Bergmann, Martin

    2007-01-01

    The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO−/− mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis. PMID:17591950

  15. Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

    PubMed Central

    Choi, Jin-Hwa; Nguyen, Minh-Phuong; Lee, Dongjin; Oh, Goo-Taeg; Lee, You-Mie

    2014-01-01

    Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT+/− EPCs. In AGT+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis. PMID:24938229

  16. Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development

    PubMed Central

    Wang, Lu; Wang, Yan; Zhou, Shimeng; Yang, Liukun; Shi, Qixin; Li, Yujiao; Zhang, Kun; Yang, Le; Zhao, Minggao; Yang, Qi

    2016-01-01

    Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment. PMID:27517961

  17. Kv4.2 knockout mice have hippocampal-dependent learning and memory deficits.

    PubMed

    Lugo, Joaquin N; Brewster, Amy L; Spencer, Corinne M; Anderson, Anne E

    2012-05-01

    Kv4.2 channels contribute to the transient, outward K(+) current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit in the learning phase of the Morris water maze (MWM) and significant impairment in the probe trial compared with wild type (WT). Kv4.2 KO mice also demonstrated a specific deficit in contextual learning in the fear-conditioning test, without impairment in the conditioned stimulus or new context condition. Kv4.2 KO mice had normal activity, anxiety levels, and prepulse inhibition compared with WT mice. A compensatory increase in tonic inhibition has been previously described in hippocampal slice recordings from Kv4.2 KO mice. In an attempt to decipher whether increased tonic inhibition contributed to the learning and memory deficits in Kv4.2 KO mice, we administered picrotoxin to block GABA(A) receptors (GABA(A)R), and thereby tonic inhibition. This manipulation had no effect on behavior in the WT or KO mice. Furthermore, total protein levels of the α5 or δ GABA(A)R subunits, which contribute to tonic inhibition, were unchanged in hippocampus. Overall, our findings add to the growing body of evidence, suggesting an important role for Kv4.2 channels in hippocampal-dependent learning and memory. PMID:22505720

  18. Melting relations in the MgO-MgSiO3 system under the lower mantle conditions using a double-sided CO2 laser heated diamond anvil cell

    NASA Astrophysics Data System (ADS)

    Ohnishi, S.; Kuwayama, Y.; Inoue, T.

    2015-12-01

    Seismological observations of the ultralow-velocity zones (ULVZs) suggest the presence of partial melts above the core-mantle boundary (CMB). Knowledge of the melting relations in the lower mantle is a key to understanding the chemical differentiation at the base of the mantle. While melting relations of mantle materials at relatively low pressures (below 30 GPa) have been extensively studied using a multi-anvil apparatus (e.g. Ito et al., 2004 Phy. Earth Planet. Inter.), melting experiments at higher pressures are still limited. Only a few model compositions, such as peridotite and mid-oceanic ridge basalt (MORB), were studied under the CMB conditions using a laser-heated diamond anvil cell (LHDAC) (e.g. Fiquet et al., 2010 Science, Andrault et al., 2014 Science). Since chemical heterogeneity of both major elements (Mg, Si, Fe, Al...) and minor ones (e.g. alkalis and volatiles) should have a large effect on the melting behavior, the melting phase diagrams as a function of composition are fundamental to understand the nature of the ULVZs. For melting relations in a binary system MgO-MgSiO3, which is a major component in the lower mantle, previous experiments were performed up to only 26 GPa (Liebske and Frost, 2012 Earth Planet. Sci. Lett.). Further studies at higher pressures corresponding to the deep lower mantle conditions are required. In this study, we have determined the melting relations in the MgO-MgSiO3 system above 30 GPa using a LHDAC. Glasses of several different compositions in the MgO-MgSiO3 system (from 37 to 45 mol% SiO2) were used as starting materials. A double-sided CO2 laser heating system was used to heat the sample directly. The recovered samples were polished and analyzed by a dualbeam focused ion beam (FIB) and a field emission scanning electron microscope (FE-SEM), respectively. The eutectic compositions and the liquidus phases were determined on the basis of chemical and textural analysis of the quenched samples. Our results show that the

  19. Neurodegeneration in a Drosophila model of adrenoleukodystrophy: the roles of the Bubblegum and Double bubble acyl-CoA synthetases

    PubMed Central

    Sivachenko, Anna; Gordon, Hannah B.; Kimball, Suzanne S.; Gavin, Erin J.; Bonkowsky, Joshua L.; Letsou, Anthea

    2016-01-01

    ABSTRACT Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum (bgm) and double bubble (dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivo is causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6 (encoding a very-long-chain ACS), a human homolog of bgm and dbb. PMID:26893370

  20. Double Eye Brow: A Rare Case Report

    PubMed Central

    Kar, Sudipta; Ghosh, Chiranjit; Bazmi, Badruddin Ahamed; Sarkar, Subrata

    2013-01-01

    Eye brows are essential for esthetic and functional purposes. Various kinds of eye brows are found in human species. Protective function is one of the important functions of eye brows. Double eye brow is a very rare condition found in human. This case report describes one of the rare cases of double eye brow. PMID:24574697

  1. Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

    PubMed Central

    Zhang, Xiaowei; Bearer, Elaine L.; Boulat, Benoit; Hall, F. Scott; Uhl, George R.; Jacobs, Russell E.

    2010-01-01

    The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn2+ into the prefrontal cortex indicated that DAT KO mice have a truncated Mn2+ distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn2+ transport into more posterior midbrain nuclei and contralateral mesolimbic structures at

  2. Olmesartan vs ramipril in the treatment of hypertension and associated clinical conditions in the elderly: a reanalysis of two large double-blind, randomized studies at the light of the most recent blood pressure targets recommended by guidelines

    PubMed Central

    Omboni, Stefano; Malacco, Ettore; Mallion, Jean-Michel; Volpe, Massimo

    2015-01-01

    In this paper, we present the results of a reanalysis of the data of two large randomized, double-blind, parallel group studies with a similar design, comparing the efficacy of an angiotensin-receptor blocker (olmesartan medoxomil) with that of an angiotensin-converting enzyme inhibitor (ramipril), by applying two different blood pressure targets recently recommended by hypertension guidelines for all patients, irrespective of the presence of diabetes (<140/90 mmHg), and for elderly hypertensive patients (<150/90 mmHg). The efficacy of olmesartan was not negatively affected by age, sex, hypertension type, diabetes status or other concomitant clinical conditions, or cardiovascular risk factors. In most cases, olmesartan provided better blood pressure control than ramipril. Olmesartan was significantly more effective than ramipril in male patients, in younger patients (aged 65–69 years), in those with metabolic syndrome, obesity, dyslipidemia, preserved renal function, diastolic ± systolic hypertension, and, in general, in patients with a high or very high cardiovascular risk. Interestingly, patients previously untreated or treated with two or more antihypertensive drugs showed a significantly larger response with olmesartan than with ramipril. Thus, our results confirm the good efficacy of olmesartan in elderly hypertensives even when new blood pressure targets for antihypertensive treatment are considered. Such results may be relevant for the clinical practice, providing some hint on the possible different response of elderly hypertensive patients to two different drugs acting on the renin–angiotensin system, when patients are targeted according to the blood pressure levels recommended by recent hypertension guidelines. PMID:26491273

  3. Muscular pre-conditioning using light-emitting diode therapy (LEDT) for high-intensity exercise: a randomized double-blind placebo-controlled trial with a single elite runner.

    PubMed

    Ferraresi, Cleber; Beltrame, Thomas; Fabrizzi, Fernando; do Nascimento, Eduardo Sanches Pereira; Karsten, Marlus; Francisco, Cristina de Oliveira; Borghi-Silva, Audrey; Catai, Aparecida Maria; Cardoso, Daniel Rodrigues; Ferreira, Antonio Gilberto; Hamblin, Michael R; Bagnato, Vanderlei Salvador; Parizotto, Nivaldo Antonio

    2015-07-01

    Recently, low-level laser (light) therapy (LLLT) has been used to improve muscle performance. This study aimed to evaluate the effectiveness of near-infrared light-emitting diode therapy (LEDT) and its mechanisms of action to improve muscle performance in an elite athlete. The kinetics of oxygen uptake (VO2), blood and urine markers of muscle damage (creatine kinase--CK and alanine), and fatigue (lactate) were analyzed. Additionally, some metabolic parameters were assessed in urine using proton nuclear magnetic resonance spectroscopy ((1)H NMR). A LED cluster with 50 LEDs (λ = 850 nm; 50 mW 15 s; 37.5 J) was applied on legs, arms and trunk muscles of a single runner athlete 5 min before a high-intense constant workload running exercise on treadmill. The athlete received either Placebo-1-LEDT; Placebo-2-LEDT; or Effective-LEDT in a randomized double-blind placebo-controlled trial with washout period of 7 d between each test. LEDT improved the speed of the muscular VO2 adaptation (∼-9 s), decreased O2 deficit (∼-10 L), increased the VO2 from the slow component phase (∼+348 ml min(-1)), and increased the time limit of exercise (∼+589 s). LEDT decreased blood and urine markers of muscle damage and fatigue (CK, alanine and lactate levels). The results suggest that a muscular pre-conditioning regimen using LEDT before intense exercises could modulate metabolic and renal function to achieve better performance. PMID:25585514

  4. Impact of a trace element supplementation programme on health and performance of cross-breed (Bos indicus x Bos taurus) dairy cattle under tropical farming conditions: a double-blinded randomized field trial.

    PubMed

    Dermauw, V; Dierenfeld, E; Du Laing, G; Buyse, J; Brochier, B; Van Gucht, S; Duchateau, L; Janssens, G P J

    2015-06-01

    Small-scale urban dairy farms (n = 16) in and around Jimma, Ethiopia with cross-bred (Bos indicus × Bos taurus) cows were enrolled in a double-blinded intervention study to investigate the effect of a trace element supplementation programme on trace element status and milk concentrations as well as performance [body condition score (BCS), milk yield, leptin], milk composition, antioxidant status (ferric-reducing ability of plasma (FRAP), thiobarbituric acid-reactive substances (TBARS)], blood biochemistry, serum proteins and immune response (antibody titre upon rabies vaccination). The farms were allocated to a (1) placebo or (2) Cu, Zn, Se, Co and I supplementation treatment for 150 d. On days 0 and 120, four lactating cows per farm were sampled for milk and plasma, and on day 150 for serum, following primo-vaccination. Cu deficiency was present in 17% and marginal Se deficiency in 30% of initially sampled cows, while no Zn shortage was detected. Over 120 days, trace element supplementation caused a bigger increase in plasma Se and Cu concentrations, but also a larger decrease of plasma Fe concentrations. A larger increase in milk Se concentrations was observed in the supplemented group, whereas none of the other elements were affected. BCS decreased more over time in the supplemented group. None of the other parameters of performance and antioxidant status nor milk composition or blood biochemistry was affected by treatment. Antibody response to rabies vaccination did not differ between groups, whereas α1-globulins tended to be lower and β-globulins tended to be higher in the supplemented group. In conclusion, despite improved Cu and Se status and Se concentrations in milk, cows on tropical urban dairy farms did not seem to benefit from trace element supplementation, with respect to the parameters investigated. PMID:24990384

  5. Impaired Social Behavior in 5-HT3A Receptor Knockout Mice

    PubMed Central

    Smit-Rigter, Laura A.; Wadman, Wytse J.; van Hooft, Johannes A.

    2010-01-01

    The 5-HT3 receptor is a ligand-gated ion channel expressed on interneurons throughout the brain. So far, analysis of the 5-HT3A knockout mouse revealed changes in nociceptive processing and a reduction in anxiety related behavior. Recently, it was shown that the 5-HT3 receptor is also expressed on Cajal-Retzius cells which play a key role in cortical development and that knockout mice lacking this receptor showed aberrant growth of the dendritic tree of cortical layer II/III pyramidal neurons. Other mouse models in which serotonergic signaling was disrupted during development showed similar morphological changes in the cortex, and in addition, also deficits in social behavior. Here, we subjected male and female 5-HT3A knockout mice and their non-transgenic littermates to several tests of social behavior. We found that 5-HT3A knockout mice display impaired social communication in the social transmission of food preference task. Interestingly, we showed that in the social interaction test only female 5-HT3A knockout mice spent less time in reciprocal social interaction starting after 5 min of testing. Moreover, we observed differences in preference for social novelty for male and female 5-HT3A knockout mice during the social approach test. However, no changes in olfaction, exploratory activity and anxiety were detected. These results indicate that the 5-HT3A knockout mouse displays impaired social behavior with specific changes in males and females, reminiscent to other mouse models in which serotonergic signaling is disturbed in the developing brain. PMID:21103015

  6. Double pituitary adenomas.

    PubMed

    Iacovazzo, D; Bianchi, A; Lugli, F; Milardi, D; Giampietro, A; Lucci-Cordisco, E; Doglietto, F; Lauriola, L; De Marinis, L

    2013-04-01

    Double pituitary adenomas represent up to 2.6 % of pituitary adenomas in large surgical series and up to 3.3 % of patients with Cushing's disease have been found to have double or multiple pituitary adenomas. We report the case of a 60-year-old male patient whose medical history began in 2002 with erectile dysfunction; hyperprolactinemia was found and MRI showed a 6-mm area of delayed enhancement in the lateral portion of the right pituitary lobe. Treatment with cabergoline was started with normalization of prolactin levels; the following MRI, performed in 2005 and 2008, showed shrinkage of the pituitary lesion. In 2005, the patient began to manifest weight gain, hypertension, and facial plethora, but no further evaluations were done. In January 2010, the patient came to our attention and underwent multiple tests that suggested Cushing's disease. A new MRI was negative. Bilateral inferior petrosal sinus sampling showed significant pituitary-to-peripheral ratio and, in May 2010, the patient underwent exploratory pituitary surgery with evidence of a 1-2-mm white-coloured midline area compatible with pituitary adenoma that was surgically removed. Post-operatively, the patient's clinical conditions improved with onset of secondary hypoadrenalism. The histologic examination confirmed a pituitary adenoma (immunostaining was found to be positive for ACTH and negative for prolactin). We report the case of an ACTH-producing microadenoma metachronous to a prolactin secreting microadenoma although not confirmed histologically, shrunk by medical treatment. A review of data in the literature regarding double or multiple pituitary adenomas has also been done. PMID:23325364

  7. Phenotypic characterization of nonsocial behavioral impairment in neurexin 1α knockout rats.

    PubMed

    Esclassan, Frederic; Francois, Jennifer; Phillips, Keith G; Loomis, Sally; Gilmour, Gary

    2015-02-01

    Neurexins are neuronal presynaptic proteins that play a key role in mediation of synapse formation. Heterozygous partial deletions in the neurexin-1 gene (NRXN1, 2p16.3) have been observed in autism spectrum disorder (ASD) patients. NRXN1-α knockout (KO) mice present behavioral impairments that resemble some of the core ASD symptoms of social impairment and inflexibility/stereotypy. At present, a thorough assessment of cognitive function has yet to be completed. Rats, containing a biallelic deletion of the NRNX1-α gene on a Sprague Dawley background were compared to littermate wild types across a range of tasks designed to test functional domains disrupted in ASD and other neurodevelopmental disorders, including sensory perception (prepulse inhibition), attention (latent inhibition), associative learning (instrumental and Pavlovian conditioning), and memory (rewarded alternation T maze and spatial discrimination). NRXN1α KO rats were found to present with large and persistent nonsocial deficits, including hyperactivity, deficits in simple instrumental learning, latent inhibition, and spatial-dependent learning. No deficit in sensorimotor gating was observed, despite the presence of an exaggerated startle response. Although KO animals were also able to learn a simple Pavlovian conditioning discrimination, they did display impaired latent inhibition. The presence of pronounced impairments in several domains in NRXN1α KO rats clearly suggests that nonsocial cognitive deficits can also be measured in an animal model of ASD. Further exploration of those deficits, both clinically and preclinically, as planned in the Innovative Medicines Initiative's European Autism Interventions: A Multicenter Study for Developing New Medications program, may help to better understand the brain circuitry involved in ASD and therefore open new avenues to advance novel therapies. PMID:25420124

  8. Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice

    PubMed Central

    Powell, David R.; Gay, Jason P.; Wilganowski, Nathaniel; Doree, Deon; Savelieva, Katerina V.; Lanthorn, Thomas H.; Read, Robert; Vogel, Peter; Hansen, Gwenn M.; Brommage, Robert; Ding, Zhi-Ming; Desai, Urvi; Zambrowicz, Brian

    2015-01-01

    After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side

  9. Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice.

    PubMed

    Powell, David R; Gay, Jason P; Wilganowski, Nathaniel; Doree, Deon; Savelieva, Katerina V; Lanthorn, Thomas H; Read, Robert; Vogel, Peter; Hansen, Gwenn M; Brommage, Robert; Ding, Zhi-Ming; Desai, Urvi; Zambrowicz, Brian

    2015-01-01

    After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side

  10. Outer-Shell Double Photoionization of CH4 and CH2Cl2 Molecules

    NASA Astrophysics Data System (ADS)

    Alcantara, K. F.; Gomes, A. H. A.; Sigaud, L.; Wolf, W.; Santos, A. C. F.

    In this work the roles of the shake-off and knockout processes in the double photoionization of the CH2Cl2 and CH4 molecules have been studied. The probabilities for both mechanisms accompanying valence-shell photoionization have been estimated as a function of incident photon energy using Samson's (1990) and Thomas's (1994) models, respectively. The experimental results are in qualitative accord with the models.

  11. Double field theory inspired cosmology

    SciTech Connect

    Wu, Houwen; Yang, Haitang E-mail: hyanga@scu.edu.cn

    2014-07-01

    Double field theory proposes a generalized spacetime action possessing manifest T-duality on the level of component fields. We calculate the cosmological solutions of double field theory with vanishing Kalb-Ramond field. It turns out that double field theory provides a more consistent way to construct cosmological solutions than the standard string cosmology. We construct solutions for vanishing and non-vanishing symmetry preserving dilaton potentials. The solutions assemble the pre- and post-big bang evolutions in one single line element. Our results show a smooth evolution from an anisotropic early stage to an isotropic phase without any special initial conditions in contrast to previous models. In addition, we demonstrate that the contraction of the dual space automatically leads to both an inflation phase and a decelerated expansion of the ordinary space during different evolution stages.

  12. Monoglyceride lipase deficiency modulates endocannabinoid signaling and improves plaque stability in ApoE-knockout mice

    PubMed Central

    Vujic, Nemanja; Schlager, Stefanie; Eichmann, Thomas O.; Madreiter-Sokolowski, Corina T.; Goeritzer, Madeleine; Rainer, Silvia; Schauer, Silvia; Rosenberger, Angelika; Woelfler, Albert; Doddapattar, Prakash; Zimmermann, Robert; Hoefler, Gerald; Lass, Achim; Graier, Wolfgang F.; Radovic, Branislav; Kratky, Dagmar

    2016-01-01

    Background and aims Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading monoglyceride (MG) to glycerol and fatty acid. MGL also hydrolyzes and thereby deactivates 2-arachidonoyl glycerol (2-AG), the most abundant endocannabinoid in the mammalian system. 2-AG acts as full agonist on cannabinoid receptor type 1 (CB1R) and CB2R, which are mainly expressed in brain and immune cells, respectively. Thus, we speculated that in the absence of MGL, increased 2-AG concentrations mediate CB2R signaling in immune cells to modulate inflammatory responses, thereby affecting the development of atherosclerosis. Methods and results We generated apolipoprotein E (ApoE)/MGL double-knockout (DKO) mice and challenged them with Western-type diet for 9 weeks. Despite systemically increased 2-AG concentrations in DKO mice, CB2R-mediated signaling remains fully functional, arguing against CB2R desensitization. We found increased plaque formation in both en face aortae (1.3-fold, p = 0.028) and aortic valve sections (1.5-fold, p = 0.0010) in DKO mice. Interestingly, DKO mice also presented reduced lipid (12%, p = 0.031) and macrophage content (18%, p = 0.061), elevated intraplaque smooth muscle staining (1.4-fold, p = 0.016) and thicker fibrous caps (1.8-fold, p = 0.0032), together with a higher ratio of collagen to necrotic core area (2.5-fold, p = 0.0003) and expanded collagen content (1.6-fold, p = 0.0007), which suggest formation of less vulnerable atherosclerotic plaques. Treatment with a CB2R inverse agonist prevents these effects in DKO mice, demonstrating that the observed plaque phenotype in DKO mice originates from CB2R activation. Conclusion Loss of MGL modulates endocannabinoid signaling in CB2R-expressing cells, which concomitantly affects the pathogenesis of atherosclerosis. We conclude that despite larger lesion size loss of MGL improves atherosclerotic plaque stability. Thus, pharmacological MGL inhibition may be a novel intervention to reduce

  13. Gene Knockout Identification Using an Extension of Bees Hill Flux Balance Analysis

    PubMed Central

    Choon, Yee Wen; Mohamad, Mohd Saberi; Deris, Safaai; Chong, Chuii Khim; Omatu, Sigeru; Corchado, Juan Manuel

    2015-01-01

    Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA) to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes. PMID:25874200

  14. Featured Article: Accelerated decline of physical strength in peroxiredoxin-3 knockout mice.

    PubMed

    Zhang, Yong-Gang; Wang, Li; Kaifu, Tomonori; Li, Jingmin; Li, Xiaoyan; Li, Lianqin

    2016-07-01

    As a member of peroxiredoxin family, peroxiredoxin-3 plays a major role in the control of mitochondrial level of reactive oxygen species. During the breeding of experimental mice, we noticed that the peroxiredoxin-3 knockout mice were listless with aging. In the present study, we compared the swimming exercise performance and oxidative status between peroxiredoxin-3 knockout mice (n = 15) and wild-type littermates (n = 15). At the age of 10 months, the physical strength of peroxiredoxin-3 knockout mice was much lower than the wild-type littermates. Increased oxidative damage and decreased mitochondrial DNA copy number of the animal skeletal muscles were observed in peroxiredoxin-3 knockout mice as compared to that in the wild-type littermates. In addition, we found increased apoptotic cells in the brains of peroxiredoxin-3 knockout mice. Our results suggest that the deficiency of peroxiredoxin-3 induces accelerated oxidative stress and mitochondrial impairment, resulting in the decrease of energy supply and cellular activities. Peroxiredoxin-3 might be involved in the inhibition of aging process. PMID:27037278

  15. Glutamate transporter type 3 knockout leads to decreased heart rate possibly via parasympathetic mechanism.

    PubMed

    Deng, Jiao; Li, Jiejie; Li, Liaoliao; Feng, Chenzhuo; Xiong, Lize; Zuo, Zhiyi

    2013-08-01

    Parasympathetic tone is a dominant neural regulator for basal heart rate. Glutamate transporters (EAAT) via their glutamate uptake functions regulate glutamate neurotransmission in the central nervous system. We showed that EAAT type 3 (EAAT3) knockout mice had a slower heart rate than wild-type mice when they were anesthetized. We design this study to determine whether non-anesthetized EAAT3 knockout mice have a slower heart rate and, if so, what may be the mechanism for this effect. Young adult EAAT3 knockout mice had slower heart rates than those of their littermate wild-type mice no matter whether they were awake or anesthetized. This difference was abolished by atropine, a parasympatholytic drug. Carbamylcholine chloride, a parasympathomimetic drug, equally effectively reduced the heart rates of wild-type and EAAT3 knockout mice. Positive immunostaining for EAAT3 was found in the area of nuclei deriving fibers for vagus nerve. There was no positive staining for the EAATs in the sinoatrial node. These results suggest that EAAT3 knockout mice have a slower heart rate at rest. This effect may be caused by an increased parasympathetic tone possibly due to increased glutamate neurotransmission in the central nervous system. These findings indicate that regulation of heart rate, a vital sign, is one of the EAAT biological functions. PMID:23361868

  16. The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice

    PubMed Central

    2010-01-01

    Background The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them. Results VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected. Conclusion VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes. PMID:21190592

  17. A double-double/double-single computation package

    SciTech Connect

    Bailey, David H.

    2004-12-01

    The DDFUNIDSFUN software permits a new or existing Fortran-90 program to utilize double-double precision (approx. 31 digits) or double-single precision (approx. 14 digits) arithmetic. Double-double precision is required by a rapidly expandirtg body of scientific computations in physics and mathematics, for which the conventional 64-bit IEEE computer arithmetic (about 16 decimal digit accuracy) is not sufficient. Double-single precision permits users of systems that do not have hardware 64-bit IEEE arithmetic (such as some game systems) to perform arithmetic at a precision nearly as high as that of systems that do. Both packages run significantly faster Than using multiple precision or arbitrary precision software for this purpose. The package includes an extensive set of low-level routines to perform high-precision arithmetic, including routines to calculate various algebraic and transcendental functions, such as square roots, sin, ccc, exp, log and others. In addition, the package includes high-level translation facilities, so that Fortran programs can utilize these facilities by making only a few changes to conventional Fortran programs. In most cases, the only changes that are required are to change the type statements of variables that one wishes to be treated as multiple precision, plus a few other minor changes. The DDFUN package is similar in functionality to the double-double part of the GD package, which was previously written at LBNL. However, the DDFUN package is written exclusively in Fortran-90, thus avoidIng difficulties that some users experience when using GD, which includes both Fortran-90 and C++ code.

  18. A double-double/double-single computation package

    Energy Science and Technology Software Center (ESTSC)

    2004-12-01

    The DDFUNIDSFUN software permits a new or existing Fortran-90 program to utilize double-double precision (approx. 31 digits) or double-single precision (approx. 14 digits) arithmetic. Double-double precision is required by a rapidly expandirtg body of scientific computations in physics and mathematics, for which the conventional 64-bit IEEE computer arithmetic (about 16 decimal digit accuracy) is not sufficient. Double-single precision permits users of systems that do not have hardware 64-bit IEEE arithmetic (such as some game systems)more » to perform arithmetic at a precision nearly as high as that of systems that do. Both packages run significantly faster Than using multiple precision or arbitrary precision software for this purpose. The package includes an extensive set of low-level routines to perform high-precision arithmetic, including routines to calculate various algebraic and transcendental functions, such as square roots, sin, ccc, exp, log and others. In addition, the package includes high-level translation facilities, so that Fortran programs can utilize these facilities by making only a few changes to conventional Fortran programs. In most cases, the only changes that are required are to change the type statements of variables that one wishes to be treated as multiple precision, plus a few other minor changes. The DDFUN package is similar in functionality to the double-double part of the GD package, which was previously written at LBNL. However, the DDFUN package is written exclusively in Fortran-90, thus avoidIng difficulties that some users experience when using GD, which includes both Fortran-90 and C++ code.« less

  19. The Cambridge Double Star Atlas

    NASA Astrophysics Data System (ADS)

    MacEvoy, Bruce; Tirion, Wil

    2015-12-01

    Preface; What are double stars?; The binary orbit; Double star dynamics; Stellar mass and the binary life cycle; The double star population; Detecting double stars; Double star catalogs; Telescope optics; Preparing to observe; Helpful accessories; Viewing challenges; Next steps; Appendices: target list; Useful formulas; Double star orbits; Double star catalogs; The Greek alphabet.

  20. Attenuation of experimental colitis in glutathione peroxidase 1 and catalase double knockout mice through enhancing regulatory T cell function.

    PubMed

    Kim, Hyung-Ran; Lee, Anbok; Choi, Eun-Jeong; Kie, Jeong-Hae; Lim, Woosung; Lee, Hyeon Kook; Moon, Byung-In; Seoh, Ju-Young

    2014-01-01

    Reactive oxygen species (ROS) have been implicated in the progression of inflammatory diseases including inflammatory bowel diseases (IBD). Meanwhile, several studies suggested the protective role of ROS in immune-mediated inflammatory diseases, and it was recently reported that dextran sodium sulfate (DSS)-induced colitis was attenuated in mice with an elevated level of ROS due to deficiency of peroxiredoxin II. Regulatory T cells (Tregs) are critical in the prevention of IBD and Treg function was reported to be closely associated with ROS level, but it has been investigated only in lowered levels of ROS so far. In the present study, in order to clarify the relationship between ROS level and Treg function, and their role in the pathogenesis of IBD, we investigated mice with an elevated level of ROS due to deficiency of both glutathione peroxidase (GPx)-1 and catalase (Cat) for the susceptibility of DSS-induced colitis in association with Treg function. The results showed that DSS-induced colitis was attenuated and Tregs were hyperfunctional in GPx1-/- × Cat-/- mice. In vivo administration of N-acetylcysteine (NAC) aggravated DSS-induced colitis and decreased Treg function to the level comparable to WT mice. Attenuated Th17 cell differentiation from naïve CD4+ cells as well as impaired production of IL-6 and IL-17A by splenocytes upon stimulation suggested anti-inflammatory tendency of GPx1-/- × Cat-/- mice. Suppression of Stat3 activation in association with enhancement of indoleamine 2,3-dioxygenase and FoxP3 expression might be involved in the immunosuppressive mechanism of GPx1-/- × Cat-/- mice. Taken together, it is implied that ROS level is critical in the regulation of Treg function, and IBD may be attenuated in appropriately elevated levels of ROS. PMID:24743300

  1. Thalidomide influences atherogenesis in aortas of ApoE(-/-)/LDLR (-/-) double knockout mice: a nano-CT study.

    PubMed

    Kampschulte, Marian; Gunkel, Irina; Stieger, Philipp; Sedding, Daniel G; Brinkmann, Anne; Ritman, Erik L; Krombach, Gabriele A; Langheinrich, Alexander C

    2014-04-01

    Plaque progression in atherosclerosis is closely connected to angiogenesis due to vasa vasorum (VV) growth. Objective of this study was to determine the unknown long-term effect of thalidomide on adventitial VV neovascularization and plaque progression using nano-focussed computed tomography (nano-CT). Proliferation and migration assays in human coronary artery endothelial cells (HCAEC) measured number of viable cells after incubation with thalidomide. Male ApoE(-/-)/LDLR(-/-) (AL) mice (n = 5) received a thalidomide containing western diet (WD) over 29 weeks. Another five male AL mice (WD without thalidomide) served as control group. Descending aortas were scanned with nano-CT at (1.5 μm)(3) isotropic voxel size. Number and area of adventitial VV as well as plaque cross sectional area were measured. Results were complemented by histology. Thalidomide inhibited proliferation and migration of HCAEC dose-dependently. VV neovascularization decreased in number per cross section (7.66 ± 0.301 vs. 8.62 ± 0.164, p < 0.001) and in cross sectional area (0.0183 ± 0.0011 vs. 0.0238 ± 0.0008 mm(2), p < 0.001). Cross sectional area of plaque decreased significantly when treated with thalidomide (0.57 ± 0.0187 vs. 0.803 ± 0.0148 mm(2), p < 0.001). Nano-CT imaging revealed a reduced plaque growth and VV neovascularization after long-term application of thalidomide. Therefore, nano-CT can be considered as a new method to detect therapeutic effects in experimental models of atherosclerosis. PMID:24487918

  2. Quantitative X-Ray Imaging of Intraplaque Hemorrhage in Aortas of ApoE-/-/LDL-/- Double Knockout Mice

    SciTech Connect

    Langheinrich,A.; Michniewicz, A.; Sedding, D.; Lai, B.; Jorgensen, S.; Bohle, R.; Ritman, E.

    2007-01-01

    Objectives: To determine if hemorrhage into an arterial wall can be detected in CT images by virtue of the iron content. Materials and Methods: Aortas from male apoE-/-/LDL-/- mice (n = 31) were infused in situ with contrast agent, for micro-CT scanning and histology. Roentgen-opacities within the aortic walls were identified by histology and micro-x-ray fluorescence to be iron or calcium. Dual-energy scanning was performed at 2 energy levels using synchrotron-based micro-CT [(2 {mu}m)3 voxels, 16 and 20 keV] and 64-slice CT (0.4 x 0.4 x 0.6 mm voxels, 80 and 120 kVp). Results: Opacities were identified as hemorrhage-related clusters of multiple punctate deposits, containing both Fe (0.48 x 10-12 g/voxel) and Ca (3.18 x 10-2 g/voxel), or as isolated confluent accumulations of exclusively calcium. Subtraction of the dual-energy CT scans discriminated iron from calcium deposits. Conclusion: Detection and quantification of iron deposits in hemorrhaged atherosclerotic lesions is feasible by dual-energy CT imaging.

  3. Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response

    PubMed Central

    Gutin, Jenia; Sadeh, Amit; Rahat, Ayelet; Aharoni, Amir; Friedman, Nir

    2015-01-01

    Cells must quickly respond and efficiently adapt to environmental changes. The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program. The later is regulated by two transcription factors, Msn2 and Msn4, that integrate information from upstream pathways to produce fast, tunable, and robust response to different environmental changes. To understand this integration, we employed a systematic approach to genetically dissect the contribution of various cellular pathways to Msn2/4 regulation under a range of stress and growth conditions. We established a high-throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single-knockout and 1,566 double-knockout strains that carry an HSP12-GFP allele as a reporter for Msn2/4 activity. Based on the expression of this Msn2/4 reporter in five different conditions, we identified numerous genetic and epistatic interactions between different components in the network upstream to Msn2/4. Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three-way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway. PMID:26446933

  4. Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response.

    PubMed

    Gutin, Jenia; Sadeh, Amit; Rahat, Ayelet; Aharoni, Amir; Friedman, Nir

    2015-10-01

    Cells must quickly respond and efficiently adapt to environmental changes. The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program. The later is regulated by two transcription factors, Msn2 and Msn4, that integrate information from upstream pathways to produce fast, tunable, and robust response to different environmental changes. To understand this integration, we employed a systematic approach to genetically dissect the contribution of various cellular pathways to Msn2/4 regulation under a range of stress and growth conditions. We established a high-throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single-knockout and 1,566 double-knockout strains that carry an HSP12-GFP allele as a reporter for Msn2/4 activity. Based on the expression of this Msn2/4 reporter in five different conditions, we identified numerous genetic and epistatic interactions between different components in the network upstream to Msn2/4. Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three-way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway. PMID:26446933

  5. Double outlet right ventricle

    MedlinePlus

    ... medlineplus.gov/ency/article/007328.htm Double outlet right ventricle To use the sharing features on this page, please enable JavaScript. Double outlet right ventricle (DORV) is a heart disease that is ...

  6. Characterization of physiological responses to 22 gene knockouts in Escherichia coli central carbon metabolism.

    PubMed

    Long, Christopher P; Gonzalez, Jacqueline E; Sandoval, Nicholas R; Antoniewicz, Maciek R

    2016-09-01

    Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner-Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts. Analysis of collective trends and correlations in the data using principal component analysis (PCA) provide new, and sometimes surprising, insights into E. coli physiology. Additionally, by comparing the data-to-model predictions from constraint-based approaches such as FBA, MOMA and RELATCH we demonstrate the important role of less well-understood kinetic and regulatory effects in central carbon metabolism. PMID:27212692

  7. MR histology of advanced atherosclerotic lesions of ApoE- knockout mice

    NASA Astrophysics Data System (ADS)

    Naumova, A.; Yarnykh, V.; Ferguson, M.; Rosenfeld, M.; Yuan, C.

    2016-02-01

    The purposes of this study were to examine the feasibility of determining the composition of advanced atherosclerotic plaques in fixed ApoE-knockout mice and to develop a time-efficient microimaging protocol for MR histological imaging on mice. Five formalin-fixed transgenic ApoE-knockout mice were imaged at the 9.4T Bruker BioSpec MR scanner using 3D spoiled gradient-echo sequence with an isotropic field of view of 24 mm3; TR 20.8 ms; TE 2.6 ms; flip angle 20°, resulted voxel size 47 × 63 × 94 pm3. MRI examination has shown that advanced atherosclerotic lesions of aorta, innominate and carotid arteries in ApoE-knockout mice are characterized by high calcification and presence of the large fibrofatty nodules. MRI quantification of atherosclerotic lesion components corresponded to histological assessment of plaque composition with a correlation coefficient of 0.98.

  8. Chromosome doubling method

    DOEpatents

    Kato, Akio

    2006-11-14

    The invention provides methods for chromosome doubling in plants. The technique overcomes the low yields of doubled progeny associated with the use of prior techniques for doubling chromosomes in plants such as grasses. The technique can be used in large scale applications and has been demonstrated to be highly effective in maize. Following treatment in accordance with the invention, plants remain amenable to self fertilization, thereby allowing the efficient isolation of doubled progeny plants.

  9. Effect of knockout of α2δ-1 on action potentials in mouse sensory neurons.

    PubMed

    Margas, Wojciech; Ferron, Laurent; Nieto-Rostro, Manuela; Schwartz, Arnold; Dolphin, Annette C

    2016-08-01

    Gene deletion of the voltage-gated calcium channel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca(2+) entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca(2+) buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca(2+)-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca(2+) elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377724

  10. Effect of knockout of α2δ-1 on action potentials in mouse sensory neurons

    PubMed Central

    Margas, Wojciech; Ferron, Laurent; Nieto-Rostro, Manuela; Schwartz, Arnold; Dolphin, Annette C.

    2016-01-01

    Gene deletion of the voltage-gated calcium channel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca2+ entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca2+ buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca2+-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca2+ elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377724

  11. CD47 gene knockout protects against transient focal cerebral ischemia in mice.

    PubMed

    Jin, Guang; Tsuji, Kiyoshi; Xing, Changhong; Yang, Yong-Guang; Wang, Xiaoying; Lo, Eng H

    2009-05-01

    CD47 is a cell surface glycoprotein that helps mediate neutrophil transmigration across blood vessels. The present study was performed to determine whether absence of the CD47 gene decreases focal ischemic brain damage. Mice were subjected to 90 min middle cerebral artery occlusion. CD47 knockout mice were compared against matching wildtype mice. CD47 expression was checked by Western blotting. Infarct volume and ischemic brain swelling were quantified with cresyl violet-stained brain sections at 24 and 72 h after ischemia. The tight junction protein claudin-5 was detected by imunohistochemistry. Two surrogate markers of neuroinflammation, brain levels of matrix metalloproteinase-9 (MMP-9) and infiltration of neutrophils, were assessed by immunohistochemistry. Western blots confirmed that CD47 was absent in knockout brains. Ischemia did not appear to upregulate total brain levels of CD47 in WT mice. In CD47 knockout mice, infarct volumes were reduced at 24 and 72 h after ischemia, and hemispheric swelling was decreased at 72 h. Loss of claudin-5 was observed in ischemic WT brain. This effect was ameliorated in CD47 knockout brains. Extravasation of neutrophils into the brain parenchyma was significantly reduced in CD47 knockout mice compared to wildtype mice. MMP-9 appeared to be upregulated in microvessels within ischemic brain. MMP-9 levels were markedly lower in CD47 knockout brains compared to wildtype brains. We conclude that CD47 is broadly involved in neuroinflammation, and this integrin-associated-protein plays a role in promoting MMP-9 upregulaton, neutrophil extravasation, brain swelling and progression of acute ischemic brain injury. PMID:19233173

  12. Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.

    PubMed

    Williams, Michael R; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T; Luikart, Bryan W

    2015-01-21

    Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either "birthdate" or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. PMID:25609613

  13. Distortion effects on the neutron knockout from exotic nuclei in the collision with a proton target

    NASA Astrophysics Data System (ADS)

    Cravo, E.; Crespo, R.; Deltuva, A.

    2016-05-01

    Background: Reaction theory plays a major role in the interpretation of experimental data and one needs to identify and include accurately all the relevant dynamical effects in order to extract reliable structure information. The knockout of a nucleon (neutron/proton) from a high energy exotic nucleus projectile colliding with a proton target allows to get insight on the structure of its valence and inner shells. Purpose: We aim to clarify the role of the distortion on the calculated observables for nucleon knockout, in particular, the dependence of the calculated observables on the binding energy ɛb and angular momentum L of the knockout particle, and on the mass of the projectile core, Ac. We consider mainly the knockout of a neutron that may be either in the valence or in the inner shell of the projectile nucleus. Method: Exact three-body Faddeev/Alt-Grassberger-Sandhas (Faddeev/AGS) calculations are performed for the nucleon knockout from stable and exotic nuclei in the collision of 420 MeV/u projectile beams with a proton target. Results are compared with plane-wave impulse approximation (PWIA) calculations. Results: The Faddeev/AGS formalism accurately predicts: (i) a systematic nearly logarithmic dependence of the distortion parameter on the separation energy; (ii) roughly linear dependence of the ratio of the full to the PWIA cross section on the asymmetry parameter; (iii) a distinct behavior between the calculated transverse core momentum distribution from the PWIA and full Faddeev/AGS exact approach which indicates that distortion effects do not modify fully exclusive observables through a common renormalization factor. Conclusions: To extract structure information on deeper shells one needs to include distortion effects accurately. A systematic analysis enables to estimate the total cross section for knockout of a nucleon from a given shell of nuclei at/away the stability line of the nuclear landscape. The comparison with experimental results may

  14. Alterations of Colonic Contractility in an Interleukin-10 Knockout Mouse Model of Inflammatory Bowel Disease

    PubMed Central

    Park, Jae Hyung; Kwon, Joong Goo; Kim, Sun Joo; Song, Dae Kyu; Lee, Seok Guen; Kim, Eun Su; Cho, Kwang Bum; Jang, Byung Ik; Kim, Dae Hwan; Sin, Jeong-Im; Kim, Tae Wan; Song, In Hwan; Park, Kyung Sik

    2015-01-01

    Background/Aims Inflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10−/−) mice. Methods Prepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining. Results The spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10−/− mice compared to IL-10+/+ mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10−/− mice compared to IL-10+/+ mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10−/− mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10−/− mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased. Conclusions These results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon. PMID:25537671

  15. 5’-ectonucleotidase knockout mice lack non-REM sleep responses to sleep deprivation

    PubMed Central

    Zielinski, Mark R.; Taishi, Ping; Clinton, James M.; Krueger, James M.

    2012-01-01

    Adenosine and extracellular adenosine triphosphate (ATP) have multiple physiological central nervous system (CNS) actions including regulation of cerebral blood flow, inflammation, and sleep. However, their exact sleep regulatory mechanisms remain unknown. Extracellular ATP and adenosine diphosphate (ADP) are converted to adenosine monophosphate (AMP) by the enzyme ectonucleoside triphosphate diphosphohydrolase 1, also known as CD39, and extracellular AMP is in turn converted to adenosine by the 5’-ectonuleotidase enzyme CD73. We investigated the role of CD73 in sleep regulation. Duration of spontaneous non-rapid eye movement sleep (NREMS) was greater in CD73 knockout (KO) mice compared to C57BL/6 controls whether determined in our laboratory or by others. After sleep deprivation (SD), NREMS was enhanced in controls but not CD73 KO mice. Interleukin-1 beta (IL1β) enhanced NREMS in both strains indicating that the CD73 KO mice were capable of sleep responses. Electroencephalographic (EEG) power spectra during NREMS in the 1.0–2.5 Hz frequency range was significantly enhanced after SD in both CD73 KO and WT mice; the increases were significantly greater in the WT mice vs. CD73 KO mice. Rapid eye movement sleep did not differ between strains in any of the experimental conditions. With the exception of CD73 mRNA, the effects of SD on various adenosine-related mRNAs were small and similar in both strains. These data suggest that sleep is regulated, in part, by extracellular adenosine derived from the actions of CD73. PMID:22540145

  16. Impaired striatum-dependent behavior in GASP-1-knock-out mice.

    PubMed

    Mathis, C; Bott, J-B; Candusso, M-P; Simonin, F; Cassel, J-C

    2011-04-01

    G protein-coupled receptor (GPCR) associated sorting protein-1 (GASP-1) is suspected to play a key role in recycling and degradation of several GPCRs. In a previous study, we have shown that GASP-1-knock-out (GASP-1-KO) mice displayed deficits in acquiring a cocaine self-administration task, associated with an exacerbated down-regulation of striatal dopaminergic and cholinergic receptors. Among several possibilities, GASP-1 deficiency could have impaired memory processes underlying the acquisition of the operant conditioning task. Therefore, the present study investigated cognitive performances of GASP-1-KO mice and their wild-type littermates (WT) in a broad variety of memory tasks. Consistent with a deficit in procedural memory, GASP-1-KO mice showed delayed acquisition of a food-reinforced bar-press task. During water-maze training in hidden- or visible-platform paradigms, mutant and WT mice acquired the tasks at the same rate. However, GASP-1 mice exhibited persistent thigmotaxic swimming, longer distance to the platform, and reduced swim speed. There was no deficit in several tasks requiring simple behavioral responses (Barnes maze, object recognition and passive avoidance tasks). Thus, the ability to acquire and/or express complex responses seems affected in GASP-1-deficient mice. Hippocampal functions were preserved, as the retention of an acquired memory in spatial tasks remained unaffected. The pattern of behavioral deficits observed in GASP-1-KO mice is coherent with current knowledge on the role of striatal GPCRs in acquisition/expression of skilled behavior and in motivation. Together with the previous findings, the so far established phenotype of GASP-1-KO mice makes them a potentially exciting tool to study striatal functions. PMID:21091868

  17. Gut Microbiota Is a Key Modulator of Insulin Resistance in TLR 2 Knockout Mice

    PubMed Central

    Caricilli, Andréa M.; Picardi, Paty K.; de Abreu, Lélia L.; Ueno, Mirian; Prada, Patrícia O.; Ropelle, Eduardo R.; Hirabara, Sandro Massao; Castoldi, Ângela; Vieira, Pedro; Camara, Niels O. S.; Curi, Rui; Carvalheira, José B.; Saad, Mário J. A.

    2011-01-01

    Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other

  18. The in vivo respiratory phenotype of the adenosine A1 receptor knockout mouse.

    PubMed

    Heitzmann, Dirk; Buehler, Philipp; Schweda, Frank; Georgieff, Michael; Warth, Richard; Thomas, Joerg

    2016-02-01

    The nucleoside adenosine has been implicated in the regulation of respiration, especially during hypoxia in the newborn. In this study the role of adenosine A1 receptors for the control of respiration was investigated in vivo. To this end, respiration of unrestrained adult and neonatal adenosine A1 receptor knockout mice (A1R(-/-)) was measured in a plethysmographic device. Under control conditions (21% O2) and mild hypoxia (12-15% O2) no difference of respiratory parameters was observed between adult wildtype (A1R(+/+)) and A1R(-/-) mice. Under more severe hypoxia (6-10% O2) A1R(+/+) mice showed, after a transient increase of respiration, a decrease of respiration frequency (fR) and tidal volume (VT) leading to a decrease of minute volume (MV). This depression of respiration during severe hypoxia was absent in A1R(-/-) mice which displayed a stimulated respiration as indicated by the enhancement of MV by some 50-60%. During hypercapnia-hyperoxia (3-10% CO2/97-90 % O2), no obvious differences in respiration of A1R(-/-) and A1R(+/+) was observed. In neonatal mice, the respiratory response to hypoxia was surprisingly similar in both genotypes. However, neonatal A1R(-/-) mice appeared to have more frequently periods of apnea during hypoxia and in the post-hypoxic control period. In conclusion, these data indicate that the adenosine A1 receptor is an important molecular component mediating hypoxic depression in adult mice and it appears to stabilize respiration of neonatal mice. PMID:26593641

  19. Effect of knockout serum replacement supplementation to culture medium on porcine blastocyst development and piglet production.

    PubMed

    Sakurai, Masahiro; Suzuki, Chie; Yoshioka, Koji

    2015-03-01

    We have previously developed chemically defined media suitable for in vitro production (IVP) of porcine embryos and subsequently generated piglets by nonsurgical embryo transfer. In this study, to further improve the culture conditions for IVP of porcine embryos, we evaluated the effect of knockout serum replacement (KSR), a substitute for serum or albumin, on the viability and development of porcine blastocysts. The addition of 5% (v:v) KSR to porcine blastocyst medium (PBM) on Day 5 (Day 0 = IVF) significantly increased the survival and hatching rates of blastocysts and the total cell number of Day-7 blastocysts compared with those in cultures without KSR or addition of 10% fetal bovine serum. Furthermore, the number of cells in the trophectoderm of Day-6 blastocysts and the ATP content of Day-7 blastocysts cultured with 5% KSR were significantly higher than those of blastocysts cultured without KSR. The mRNA expression of a rate-limiting enzyme in β-oxidation, carnitine palmitoyltransferase 1, in Day-6 blastocysts, and a serine proteinase, urokinase-type plasminogen activator, in Day-7 blastocysts cultured in 5% KSR-PBM was significantly higher than that of blastocysts cultured in PBM alone. Four of eight recipients (50%), in which Day-5 blastocysts treated with 5% KSR were transferred nonsurgically, became pregnant. However, the efficiency of piglet production (percentage of piglets born based on the number of embryos transferred) was similar to recipients with transferred blastocysts treated without KSR. The present study demonstrated that the addition of KSR to PBM enhanced the in vitro viability of porcine blastocysts. In addition, our data suggest that KSR improved development to the hatching stage and blastocyst quality by increasing ATP content and hatching-related mRNA expression of blastocysts. PMID:25434774

  20. Effect of mineralocorticoid treatment in mice with collecting duct-specific knockout of endothelin-1.

    PubMed

    Lynch, I Jeanette; Welch, Amanda K; Gumz, Michelle L; Kohan, Donald E; Cain, Brian D; Wingo, Charles S

    2015-12-15

    Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD-specific ET-1 knockout (CD ET-1 KO) mice. Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice, and by the end of the study the CD ET-1 KO mice were substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and that lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to and escape from the chronic effects of mineralocorticoids. PMID:26400543

  1. Comprehensive behavioral study of mGluR3 knockout mice: implication in schizophrenia related endophenotypes

    PubMed Central

    2014-01-01

    Background We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved. Results We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice. Conclusions These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice. PMID:24758191

  2. Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice.

    PubMed

    Moak, Shari L; Dougan, Grace C; MarElia, Catherine B; Danse, Whitney A; Fernandez, Amanda M; Kuehl, Melanie N; Athanason, Mark G; Burkhardt, Brant R

    2014-11-01

    Pancreatic-derived factor (PANDER; also known as FAM3B) is a uniquely structured protein strongly expressed within and secreted from the endocrine pancreas. PANDER has been hypothesized to regulate fasting and fed glucose homeostasis, hepatic lipogenesis and insulin signaling, and to serve a potential role in the onset or progression of type 2 diabetes (T2D). Despite having potentially pivotal pleiotropic roles in glycemic regulation and T2D, there has been limited generation of stable animal models for the investigation of PANDER function, and there are no models on well-established genetic murine backgrounds for T2D. Our aim was to generate an enhanced murine model to further elucidate the biological function of PANDER. Therefore, a pure-bred PANDER knockout C57BL/6 (PANKO-C57) model was created and phenotypically characterized with respect to glycemic regulation and hepatic insulin signaling. The PANKO-C57 model exhibited an enhanced metabolic phenotype, particularly with regard to enhanced glucose tolerance. Male PANKO-C57 mice displayed decreased fasting plasma insulin and C-peptide levels, whereas leptin levels were increased as compared with matched C57BL/6J wild-type mice. Despite similar peripheral insulin sensitivity between both groups, hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic PKB/Akt and AMPK, along with mature SREBP-1 expression. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content as compared with wild-type C57BL/6 mice. In summary, the PANKO-C57 mouse represents a suitable model for the investigation of PANDER in multiple metabolic states and provides an additional tool to elucidate the biological function and potential role in T2D. PMID:25217499

  3. Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice

    PubMed Central

    Moak, Shari L.; Dougan, Grace C.; MarElia, Catherine B.; Danse, Whitney A.; Fernandez, Amanda M.; Kuehl, Melanie N.; Athanason, Mark G.; Burkhardt, Brant R.

    2014-01-01

    Pancreatic-derived factor (PANDER; also known as FAM3B) is a uniquely structured protein strongly expressed within and secreted from the endocrine pancreas. PANDER has been hypothesized to regulate fasting and fed glucose homeostasis, hepatic lipogenesis and insulin signaling, and to serve a potential role in the onset or progression of type 2 diabetes (T2D). Despite having potentially pivotal pleiotropic roles in glycemic regulation and T2D, there has been limited generation of stable animal models for the investigation of PANDER function, and there are no models on well-established genetic murine backgrounds for T2D. Our aim was to generate an enhanced murine model to further elucidate the biological function of PANDER. Therefore, a pure-bred PANDER knockout C57BL/6 (PANKO-C57) model was created and phenotypically characterized with respect to glycemic regulation and hepatic insulin signaling. The PANKO-C57 model exhibited an enhanced metabolic phenotype, particularly with regard to enhanced glucose tolerance. Male PANKO-C57 mice displayed decreased fasting plasma insulin and C-peptide levels, whereas leptin levels were increased as compared with matched C57BL/6J wild-type mice. Despite similar peripheral insulin sensitivity between both groups, hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic PKB/Akt and AMPK, along with mature SREBP-1 expression. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content as compared with wild-type C57BL/6 mice. In summary, the PANKO-C57 mouse represents a suitable model for the investigation of PANDER in multiple metabolic states and provides an additional tool to elucidate the biological function and potential role in T2D. PMID:25217499

  4. Alpha-asarone improves striatal cholinergic function and locomotor hyperactivity in Fmr1 knockout mice.

    PubMed

    Qiu, Guozhen; Chen, Shengqiang; Guo, Jialing; Wu, Jie; Yi, Yong-Hong

    2016-10-01

    Hyperactivity is a symptom found in several neurological and psychiatric disorders, including Fragile X syndrome (FXS). The animal model of FXS, fragile X mental retardation gene (Fmr1) knockout (KO) mouse, exhibits robust locomotor hyperactivity. Alpha (α)-asarone, a major bioactive component isolated from Acorus gramineus, has been shown in previous studies to improve various disease conditions including central nervous system disorders. In this study, we show that treatment with α-asarone alleviates locomotor hyperactivity in Fmr1 KO mice. To elucidate the mechanism underlying this improvement, we evaluated the expressions of various cholinergic markers, as well as acetylcholinesterase (AChE) activity and acetylcholine (ACh) levels, in the striatum of Fmr1 KO mice. We also analyzed the AChE-inhibitory activity of α-asarone. Striatal samples from Fmr1 KO mice showed decreased m1 muscarinic acetylcholine receptor (m1 mAChR) expression, increased AChE activity, and reduced ACh levels. Treatment with α-asarone improved m1 mAChR expression and ACh levels, and attenuated the increased AChE activity. In addition, α-asarone dose-dependently inhibited AChE activity in vitro. These results indicate that direct inhibition of AChE activity and up-regulation of m1 mAChR expression in the striatum might contribute to the beneficial effects of α-asarone on locomotor hyperactivity in Fmr1 KO mice. These findings might improve understanding of the neurobiological mechanisms responsible for locomotor hyperactivity. PMID:27316341

  5. Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors

    PubMed Central

    Sahaboglu, A; Tanimoto, N; Bolz, S; Garrido, M G; Ueffing, M; Seeliger, M W; Löwenheim, H; Ekström, P; Paquet-Durand, F

    2014-01-01

    Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD. PMID:24853412

  6. PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function

    PubMed Central

    Sahaboglu, Ayse; Tanimoto, Naoyuki; Kaur, Jasvir; Sancho-Pelluz, Javier; Huber, Gesine; Fahl, Edda; Arango-Gonzalez, Blanca; Zrenner, Eberhart; Ekström, Per; Löwenheim, Hubert; Seeliger, Mathias; Paquet-Durand, François

    2010-01-01

    Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP. PMID:21124852

  7. PROXIMAL GUT MUCOSAL EPITHELIAL HOMEOSTASIS IN AGED IL-1 TYPE I RECEPTOR KNOCKOUT MICE AFTER STARVATION

    PubMed Central

    Song, Juquan; Wolf, Steven E.; Wu, Xiao-Wu; Finnerty, Celeste C.; Herndon, David N.; Jeschke, Marc G.

    2010-01-01

    Background Previous studies have shown that starvation induces small bowel atrophy, and that atrophy diminishes with aging. In this experiment, we assessed whether starvation-induced atrophy of proximal gut mucosa is associated with the Interleukin-1 receptor (IL-1R) signaling pathway in aged mice. Materials and Methods Thirty 26-month-old IL-1R knockout mice and age-matched wild-type C57BL/6 mice were randomly divided into two groups: ad libitum fed and fasted. Mice were euthanized 12 or 48 hours after starvation. The proximal small bowel was harvested for morphologic analysis. Gut epithelial cell proliferation was detected using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and apoptosis was identified using terminal deoxyuridine nick-end labeling (TUNEL) staining. Results Aged IL-1R knockout mice were larger than aged-matched wild-type mice (p<0.05). Proximal gut mucosal height and mucosal cell number were not different between aged IL-1R knockout and wild-type groups. The apoptosis index in gut epithelial cells was higher in fed IL-1R knockout versus wild-type mice (p<0.05), while no significant difference in cell proliferation between both groups. Mucosal atrophy was induced in both aged IL-1R knockout and wild-type groups by starvation (p<0.05), however, aged IL-1R knockout mice experienced greater losses in proximal gut weight, mucosal length, and corresponding cell number than did wild-type mice at the 12-hour time point (p<0.05). The apoptosis index in gut epithelial cells significantly increased in both groups after starvation (p<0.05). Starvation decreased cell proliferation in IL-1R knockout mice (p<0.05), but not in wild-type mice. Conclusions The response in aged IL-1R knockout mice differs from wild-type mice in that starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis. PMID:20605606

  8. Population of positive-parity states in {sup 53}Sc through one-proton knockout

    SciTech Connect

    McDaniel, S.; Gade, A.; Brown, B. A.; Campbell, C. M.; Cook, J. M.; Dinca, D.-C.; Glasmacher, T.; Hansen, P. G.; Terry, J. R.; Janssens, R. V. F.; Carpenter, M. P.; Zhu, S.; Bazin, D.; Mueller, W. F.; Deacon, A. N.; Freeman, S. J.; Kay, B. P.; Mantica, P. F.; Tostevin, J. A.

    2010-02-15

    The one-proton knockout reaction {sup 9}Be({sup 54}Ti,{sup 53}Sc+{gamma})X at 72 MeV/nucleon has been measured. The location of the first 3/2{sup -} state at 2110(3) keV was confirmed, and new {gamma}-ray transitions were observed at 1111(2), 1273(2), 1539(4), and 2495(5) keV. Large spectroscopic strength to excited states in {sup 53}Sc was found and attributed to the knockout of sd-shell protons.

  9. Population of positive-parity states in {sup 53}Sc through one-proton knockout.

    SciTech Connect

    McDaniel, S.; Gade, A.; Janssens, R. V. F.; Bazin, D.; Brown, B. A.; Campbell, C. M.; Carpenter, M. P.; Cook, J. M.; Deacon, A. N.; Dinca, D.-C.; Freeman, S. J.; Glasmacher, T.; Hansen, P. G.; Kay, B. P.; Mantica, P. F.; Mueller, W. F.; Terry, J. R.; Tostevin, J. A.; Zhu, S.; Physics; Michigan State Univ.; Univ. of Manchester; Univ. of Surrey

    2010-02-01

    The one-proton knockout reaction {sup 9}Be({sup 54}Ti,{sup 53}Sc+{gamma})X at 72 MeV/nucleon has been measured. The location of the first 3/2{sup -} state at 2110(3) keV was confirmed, and new {gamma}-ray transitions were observed at 1111(2), 1273(2), 1539(4), and 2495(5) keV. Large spectroscopic strength to excited states in {sup 53}Sc was found and attributed to the knockout of sd-shell protons.

  10. Cross sections for one-neutron knock-out from 37Ca at intermediate energy

    NASA Astrophysics Data System (ADS)

    Bürger, A.; Azaiez, F.; Algora, A.; Al-Khatib, A.; Bastin, B.; Benzoni, G.; Borcea, R.; Bourgeois, C.; Bringel, P.; Clément, E.; Dalouzy, J.-C.; Dlouhý, Z.; Dombrádi, Z.; Drouart, A.; Engelhardt, C.; Franchoo, S.; Fülöp, Z.; Görgen, A.; Grévy, S.; Hübel, H.; Ibrahim, F.; Korten, W.; Mrázek, J.; Navin, A.; Rotaru, F.; Roussel Chomaz, P.; Saint-Laurent, M.-G.; Sletten, G.; Sohler, D.; Sorlin, O.; Stanoiu, M.; Stefan, I.; Theisen, C.; Timis, C.; Verney, D.; Williams, S.

    2012-12-01

    The cross section for the knock-out of a deeply bound valence neutron from 37Ca at an incident beam energy of 60AMeV has been measured along with momentum distributions of the residual nuclei and γ rays from the de-excitation of the first excited state in 36Ca. As for other cases of deeply bound nucleons studied using knock-out reactions, the reduction of the measured cross section compared to theoretical predictions is stronger than those observed for near-magic stable nuclei. Both the momentum distributions and the excitation energy of the first excited state in 36Ca indicate a sizable N=16 gap.

  11. Long-term effects of diazepam treatment of epileptic GABAA receptor beta3 subunit knockout mouse in early life.

    PubMed

    Liljelund, Patricia; Ferguson, Carolyn; Homanics, Gregg; Olsen, Richard W

    2005-01-01

    The knockout mouse for the beta3 subunit of the GABAA receptor exhibits spontaneous epilepsy and hyperactivity, and has been proposed as a model for the severe developmental disorder, Angelman's syndrome, which is known to be of genetic origin. We have used this mutant to test an approach of therapeutic intervention prior to seizure onset by daily injection with diazepam during either the first or second postnatal week. Results showed differences between postnatal week 1 and week 2 injections both acutely, with respect to sedative effects, and in long-term outcome, with respect to EEG and behavioral tests measured at 12-14 weeks of age. The EEG of control mice remained unaffected under all conditions, but the EEG of beta3 (-/-) injected with diazepam in week 1 was worsened, showing increased oscillatory activity at 5-6Hz, and more myoclonic jerks, particularly among males. For beta3 (-/-) injected with diazepam in week 2, the EEG was normalized in half the mice but worsened similarly to week 1 in the other half. Neonatal diazepam injection had a long-term normalizing effect on behavior of beta3 (-/-) mice injected in week 1, but diazepam treatment in week 2 did not affect the hyperactive and circling behavior characteristic of the beta3 knockout mouse. Diazepam treatment in postnatal week 2 significantly decreased anxiety in the adult beta3 group. Diazepam treatment in both postnatal weeks 1 and 2 improved the motor coordination of beta3 (-/-) on the rotarod, although performance of control mice injected with diazepam in postnatal week 2 was significantly impaired. The observed long-term outcome of neonatal diazepam injections may result from interference with developmental processes, and shows that enhancing GABAergic activity with diazepam during the period where GABA can be excitatory can produce narrow stage-related effects on brain development. PMID:16168624

  12. 15q13.3 homozygous knockout mouse model display epilepsy-, autism- and schizophrenia-related phenotypes.

    PubMed

    Forsingdal, A; Fejgin, K; Nielsen, V; Werge, T; Nielsen, J

    2016-01-01

    The 15q13.3 microdeletion syndrome is caused by a 1.5-MB hemizygous microdeletion located on 15q13.3 affecting seven genes: FAN1; MTMR10; TRPM1; miR-211; KLF13; OTUD7A; and CHRNA7. The 15q13.3 microdeletion increases the risk of intellectual disability, epilepsy, autism spectrum disorder and schizophrenia, though the clinical profile varies considerably. Two mouse models of this syndrome, with hemizygous deletion of the orthologous region in the murine genome, have recently been shown to recapitulate a number of the behavioral and physiological deficits that characterize the human condition. Still, little is known of the underlying biological mechanisms. Eleven human cases with homozygous deletion of the 15q13.3 region have been reported, all with severe functional and physiological impairments. We therefore hypothesized that a 15q13.3 homozygous knockout would confer more pronounced behavioral and physiological deficits in mice than the 15q13.3 hemizygous deletion. Here we report the characterization of a 15q13.3 knockout mouse. We observed marked deficits including altered seizure susceptibility, autistic behavior-related phenotypes, and auditory sensory processing. Several of these deficits, albeit less pronounced, were also found in the 15q13.3 hemizygous littermates indicating a gene-dosage dependency. Our findings strongly indicate that studies of the hemi- and homozygous 15q13.3 mouse strains will facilitate understanding of the biological mechanisms of severe mental disorders. PMID:27459725

  13. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis.

    PubMed

    Tu, Lan N; Morohaku, Kanako; Manna, Pulak R; Pelton, Susanne H; Butler, W Ronald; Stocco, Douglas M; Selvaraj, Vimal

    2014-10-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo(-/-)) mice. Contrary to the early report, Tspo(-/-) mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo(-/-) mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo(-/-) mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo(-/-) mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine. PMID:24936060

  14. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  15. SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS

    EPA Science Inventory

    SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS. L.F. Strader*, S.D. Perreault, J.C. Luft*, and D.J. Dix*. US EPA/ORD, Reproductive Toxicology Div., Research Triangle Park, NC
    Heat shock proteins (HSPs) protect cells from environm...

  16. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  17. Properties of the 7He ground state from 8He neutron knockout

    NASA Astrophysics Data System (ADS)

    Aksyutina, Yu.; Johansson, H. T.; Aumann, T.; Boretzky, K.; Borge, M. J. G.; Chatillon, A.; Chulkov, L. V.; Cortina-Gil, D.; Pramanik, U. Datta; Emling, H.; Forssén, C.; Fynbo, H. O. U.; Geissel, H.; Ickert, G.; Jonson, B.; Kulessa, R.; Langer, C.; Lantz, M.; LeBleis, T.; Lindahl, A. O.; Mahata, K.; Meister, M.; Münzenberg, G.; Nilsson, T.; Nyman, G.; Palit, R.; Paschalis, S.; Prokopowicz, W.; Reifarth, R.; Richter, A.; Riisager, K.; Schrieder, G.; Simon, H.; Sümmerer, K.; Tengblad, O.; Weick, H.; Zhukov, M. V.

    2009-08-01

    The unbound nucleus 7He, produced in neutron-knockout reactions with a 240 MeV/u 8He beam in a liquid-hydrogen target, has been studied in an experiment at the ALADIN-LAND setup at GSI. From an R-matrix analysis the resonance parameters for 7He as well as the spectroscopic factor for the 6He(0+) + n configuration in its ground-state have been obtained. The spectroscopic factor is 0.61 confirming that 7He is not a pure single-particle state. An analysis of 5He data from neutron-knockout reactions of 6He in a carbon target reveals the presence of an s-wave component at low energies in the α + n relative energy spectrum. A possible low-lying exited state in 7He observed in neutron knockout data from 8He in a carbon target and tentatively interpreted as a Iπ = 1 /2- state, could not be observed in the present experiment. Possible explanations of the shape difference between the 7He resonance obtained in the two knockout reactions are discussed in terms of target-dependence or different reaction mechanisms at relativistic energies.

  18. Blueberries reduce lipid peroxidation and boost antioxidant enzymes in apoe knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ApoE knockout (ApoE-/-) mice fed AIN-93G diet (CD) formulated to contain 1 % freeze-dried whole wild blueberries (CD1 percent BB) were found to have significantly less atherosclerotic lesions in aorta. Biomarkers of lipid peroxidation, including F2-isoprostanes, hydroxyoctadecadienoic acids (HODEs) ...

  19. Effects of blueberries in prevention of atherosclerosis in apoe knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ApoE knockout (ApoE-/-) mice were fed AIN-93G diet (CD) or CD formulated to contain 1% freeze-dried whole wild blueberries (CD1% BB). Mice were sacrificed after 20 weeks on the specified diet. Atherosclerotic lesions in aortic sinus were determined by staining cryosections (10 µm) with Oil Red O. Th...

  20. IdealKnock: A framework for efficiently identifying knockout strategies leading to targeted overproduction.

    PubMed

    Gu, Deqing; Zhang, Cheng; Zhou, Shengguo; Wei, Liujing; Hua, Qiang

    2016-04-01

    In recent years, computer aided redesigning methods based on genome-scale metabolic network models (GEMs) have played important roles in metabolic engineering studies; however, most of these methods are hindered by intractable computing times. In particular, methods that predict knockout strategies leading to overproduction of desired biochemical are generally unable to do high level prediction because the computational time will increase exponentially. In this study, we propose a new framework named IdealKnock, which is able to efficiently evaluate potentials of the production for different biochemical in a system by merely knocking out pathways. In addition, it is also capable of searching knockout strategies when combined with the OptKnock or OptGene framework. Furthermore, unlike other methods, IdealKnock suggests a series of mutants with targeted overproduction, which enables researchers to select the one of greatest interest for experimental validation. By testing the overproduction of a large number of native metabolites, IdealKnock showed its advantage in successfully breaking through the limitation of maximum knockout number in reasonable time and suggesting knockout strategies with better performance than other methods. In addition, gene-reaction relationship is well considered in the proposed framework. PMID:26948338

  1. Serotonin Transporter Knockout Rats Show Improved Strategy Set-Shifting and Reduced Latent Inhibition

    ERIC Educational Resources Information Center

    Nonkes, Lourens J. P.; van de Vondervoort, Ilse I. G. M.; de Leeuw, Mark J. C.; Wijlaars, Linda P.; Maes, Joseph H. R.; Homberg, Judith R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT[superscript -/-]) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting…

  2. CRISPR-Cas9-based knockout of the prion protein and its effect on the proteome.

    PubMed

    Mehrabian, Mohadeseh; Brethour, Dylan; MacIsaac, Sarah; Kim, Jin Kyu; Gunawardana, C Geeth; Wang, Hansen; Schmitt-Ulms, Gerold

    2014-01-01

    The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer's disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼ 120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton. PMID:25490046

  3. Brief Report: Altered Social Behavior in Isolation-Reared "Fmr1" Knockout Mice

    ERIC Educational Resources Information Center

    Heitzer, Andrew M.; Roth, Alexandra K.; Nawrocki, Lauren; Wrenn, Craige C.; Valdovinos, Maria G.

    2013-01-01

    Social behavior abnormalities in Fragile X syndrome (FXS) are characterized by social withdrawal, anxiety, and deficits in social cognition. To assess these deficits, a model of FXS, the "Fmr1" knockout mouse ("Fmr1" KO), has been utilized. This mouse model has a null mutation in the fragile X mental retardation 1 gene ("Fmr1") and displays…

  4. PrP{sup C} displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrP{sup C} knockout mice

    SciTech Connect

    Bertuchi, Fernanda R.; Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R.; Cerchiaro, Giselle

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer PrP{sup C} in solution acts as a radical scavenger. Black-Right-Pointing-Pointer PrP{sup C} reduces hydrogen peroxide toxicity in astrocytes. Black-Right-Pointing-Pointer Increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes. Black-Right-Pointing-Pointer PrP{sup C} prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP{sup C} protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP{sup C} in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP{sup C} decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu{sup 2+}/ascorbate/H{sub 2}O{sub 2}), which was demonstrated by approximately 70% less DMPO/OH{sup {center_dot}}. In cultured PrP{sup C}-knockout astrocytes from mice, the absence of PrP{sup C} caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H{sub 2}O{sub 2} treatment. This rapid increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP{sup C} in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  5. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

    PubMed

    Pandey, Sachchida Nand; Khawaja, Hunain; Chen, Yi-Wen

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells. PMID:26007167

  6. Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

    PubMed Central

    Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

    2014-01-01

    Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

  7. Thyrotropin receptor knockout mice: studies on immunological tolerance to a major thyroid autoantigen.

    PubMed

    Pichurin, Pavel N; Pichurina, Oxana; Marians, Russell C; Chen, Chun-Rong; Davies, Terry F; Rapoport, Basil; McLachlan, Sandra M

    2004-03-01

    Graves' disease involves a breakdown in self-tolerance to the TSH receptor (TSHR). Central T cell tolerance is established by intrathymic deletion of immature T lymphocytes that bind with high affinity to peptides from autoantigens (like the TSHR) expressed ectopically in the thymus. In TSHR-knockout mice, tolerance cannot be induced to the TSHR, which should, therefore, be a foreign antigen for these animals. To test this hypothesis, TSHR-knockout mice and wild-type controls were vaccinated (three injections) with TSHR DNA or control DNA. TSHR antibodies, measured by ELISA, binding to TSHR-expressing eukaryotic cells, and TSH binding inhibition, developed in approximately 60% of TSHR-knockout mice, not significantly different from 80% in the wild-type mice. Antibody levels were also comparable in the two groups, and both strains recognized the same immunodominant linear antibody epitope at the amino terminus of the TSHR. Splenocyte responses to TSHR protein in culture, measured as interferon-gamma production, were similar in TSHR-knockout and wild-type mice. Moreover, T cells from both strains recognized the same two epitopes from a panel of 29 synthetic peptides encompassing the TSHR ectodomain and extracellular loops. This lack of difference in immune responses in TSHR-knockout and wild-type mice is unexpected and is contrary to observations in other induced animal models of autoimmunity. The importance of our finding is that the TSHR may not be similar to other model proteins used to define the concept of central immune tolerance. PMID:14630711

  8. FMR1 Knockout mice: A model to study fragile X mental retardation

    SciTech Connect

    Oostra, B.A.; Bakker, C.E.; Reyniers, E.

    1994-09-01

    The fragile X syndrome is the most frequent form of inherited mental retardation in humans with an incidence of 1 in 1250 males and 1 in 2500 females. The clinical syndrome includes moderate to severe mental retardation, autistic behavior, macroorchidism, and facial features, such as long face with mandibular prognathism and large, everted ears. The molecular basis for this disease is a large expansion of a triplet repeat (CGG){sub n} in the 5{prime} untranslated region of the FMR1 gene. Due to this large expansion of the CGG repeat, the promoter region becomes methylated and the FMR1 gene is subsequently silenced. Hardly anything is known about the physiologic function of FMR1 and the pathologic mechanisms leading to these symptoms. Since the FMR1 gene is highly conserved in the mouse, we used the mouse to design a knockout model for the fragile X syndrome. These knockout mice lacking Fmrp have normal litter size suggesting that FMR1 is not essential in human gametogenesis and embryonic development. The knockout mice show the abnormalities also seen in the affected organs of human patients. Mutant mice show a gradual development through time of macroorchidism. In the knockout mice we observed cognitive defects in the form of deficits in learning (as shown by the hidden platform Morris water maze task) and behavioral abnormalities such as increased exploratory behavior and hyperactivity. Therefore this knockout mouse may serve as a valuable tool in studying the role of FMR1 in the fragile X syndrome and may serve as a model to elucidate the mechanisms involved in macroorchidism, abnormal behavior, and mental retardation.

  9. Impaired fear extinction as displayed by serotonin transporter knockout rats housed in open cages is disrupted by IVC cage housing.

    PubMed

    Shan, Ling; Schipper, Pieter; Nonkes, Lourens J P; Homberg, Judith R

    2014-01-01

    Anxiety disorders are influenced by both environmental and genetic factors. A well-known example for gene x environment interactions in psychiatry is the low activity (s) allelic variant of the serotonin transporter (5-HTT) promoter polymorphism (5-HTTLPR) that in the context of stress increases risk for depression and post-traumatic stress disorder (PTSD). Previously, we observed robust anxiety-related phenotypes, such as an impairment in fear extinction, in 5-HTT knockout (5-HTT-/-) versus wild-type (5-HTT+/+) rats housed in open cages. Recently, housing conditions were changed from open cages to individually ventilated cages (IVC), which are associated with a high ventilation fold and noise. This switch in housing conditions prompted an unplanned 5-HTT gene x environment interaction study in our rats. The current study shows that lifetime stress by means of IVC cage housing abolished genotype differences in fear extinction between 5-HTT-/- and 5-HTT+/+ rats. Although this effect was not attributed specifically to either the 5-HTT+/+ or the 5-HTT-/- genotype, the findings are in agreement with the modulatory role of serotonin in the processing of environmental stimuli. Our findings also underline the possibility that housing conditions confound the interpretation of anxiety-related behaviours in rodents. PMID:24658187

  10. Observing Double Stars

    NASA Astrophysics Data System (ADS)

    Genet, Russell M.; Fulton, B. J.; Bianco, Federica B.; Martinez, John; Baxter, John; Brewer, Mark; Carro, Joseph; Collins, Sarah; Estrada, Chris; Johnson, Jolyon; Salam, Akash; Wallen, Vera; Warren, Naomi; Smith, Thomas C.; Armstrong, James D.; McGaughey, Steve; Pye, John; Mohanan, Kakkala; Church, Rebecca

    2012-05-01

    Double stars have been systematically observed since William Herschel initiated his program in 1779. In 1803 he reported that, to his surprise, many of the systems he had been observing for a quarter century were gravitationally bound binary stars. In 1830 the first binary orbital solution was obtained, leading eventually to the determination of stellar masses. Double star observations have been a prolific field, with observations and discoveries - often made by students and amateurs - routinely published in a number of specialized journals such as the Journal of Double Star Observations. All published double star observations from Herschel's to the present have been incorporated in the Washington Double Star Catalog. In addition to reviewing the history of visual double stars, we discuss four observational technologies and illustrate these with our own observational results from both California and Hawaii on telescopes ranging from small SCTs to the 2-meter Faulkes Telescope North on Haleakala. Two of these technologies are visual observations aimed primarily at published "hands-on" student science education, and CCD observations of both bright and very faint doubles. The other two are recent technologies that have launched a double star renaissance. These are lucky imaging and speckle interferometry, both of which can use electron-multiplying CCD cameras to allow short (30 ms or less) exposures that are read out at high speed with very low noise. Analysis of thousands of high speed exposures allows normal seeing limitations to be overcome so very close doubles can be accurately measured.

  11. The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model.

    PubMed

    Doran, Angela; Obach, R Scott; Smith, Bill J; Hosea, Natilie A; Becker, Stacey; Callegari, Ernesto; Chen, Cuiping; Chen, Xi; Choo, Edna; Cianfrogna, Julie; Cox, Loretta M; Gibbs, John P; Gibbs, Megan A; Hatch, Heather; Hop, Cornelis E C A; Kasman, Ilana N; Laperle, Jennifer; Liu, Jianhua; Liu, Xingrong; Logman, Michael; Maclin, Debra; Nedza, Frank M; Nelson, Frederick; Olson, Emily; Rahematpura, Sandhya; Raunig, David; Rogers, Sabrinia; Schmidt, Kari; Spracklin, Douglas K; Szewc, Mark; Troutman, Matthew; Tseng, Elaine; Tu, Meihua; Van Deusen, Jeffrey W; Venkatakrishnan, Karthik; Walens, Gary; Wang, Ellen Q; Wong, Diane; Yasgar, Adam S; Zhang, Chenghong

    2005-01-01

    Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect. PMID:15502009

  12. Charge-to-Mass Dispersion Methods in Knockout-Ablation Fragmentation Models

    NASA Astrophysics Data System (ADS)

    Townsend, Lawrence; Burton, Krista; de Wet, Wouter

    2014-09-01

    Breakup of high-energy heavy ions in nuclear collisions is an important process in space radiation transport, shielding and risk assessment since the secondary particles produced by these collisions have ranges greater than their parent nucleus, and are damaging to humans and spacecraft components. This work uses a quantum-mechanical optical potential knockout-ablation model to estimate these collision cross sections in order to investigate differences in isotope and element production cross sections as a result of utilizing two different models of charge-to mass ratios for the projectile prefragments produced by the abrasion/knockout process. One model commonly used, a hypergeometric model, assumes that the distribution of abraded nucleons is completely uncorrelated. However, it permits some unrealistic distributions, such as removing all neutrons in the knockout stage, while leaving all protons intact. Another model, developed for use with a classical geometric, clean-cut abrasion model, is based upon the zero point vibrations of the giant dipole resonance of the fragmenting nucleus. In this work we compare fragment production cross section predictions using the two charge dispersion models with published experimental data. Breakup of high-energy heavy ions in nuclear collisions is an important process in space radiation transport, shielding and risk assessment since the secondary particles produced by these collisions have ranges greater than their parent nucleus, and are damaging to humans and spacecraft components. This work uses a quantum-mechanical optical potential knockout-ablation model to estimate these collision cross sections in order to investigate differences in isotope and element production cross sections as a result of utilizing two different models of charge-to mass ratios for the projectile prefragments produced by the abrasion/knockout process. One model commonly used, a hypergeometric model, assumes that the distribution of abraded nucleons is

  13. Global Gene Expression Profiling in PAI-1 Knockout Murine Heart and Kidney: Molecular Basis of Cardiac-Selective Fibrosis

    PubMed Central

    Ghosh, Asish K.; Murphy, Sheila B.; Kishore, Raj; Vaughan, Douglas E.

    2013-01-01

    Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and

  14. Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

    SciTech Connect

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; O'Roark, Erin M.; Kenyon, Nicholas J.; Last, Jerold A.

    2010-01-01

    Arginase1 and nitric oxide synthase2 (NOS2) utilize L-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N{sup o}mega-hydroxy-nor-L-arginine (nor-NOHA) significantly increased total L-arginine content in the airway compartment. We hypothesized that such an increase in L-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that L-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2

  15. Sustained delayed gastric emptying during repeated restraint stress in oxytocin knockout mice.

    PubMed

    Babygirija, R; Zheng, J; Bülbül, M; Cerjak, D; Ludwig, K; Takahashi, T

    2010-11-01

    We have recently shown that impaired gastric motility observed in acute restraint stress was restored following repeated restraint stress in mice. Repeated restraint stress up-regulates oxytocin mRNA expression and down-regulates corticotrophin-releasing factor (CRF) mRNA expression at the hypothalamus. Oxytocin knockout mice (OXT-KO) have been widely used to study the central oxytocin signalling pathways in response to various stressors. We studied the effects of acute and repeated restraint stress on solid gastric emptying and hypothalamic CRF mRNA expression in wild-type (WT) and OXT-KO mice. Heterozygous (HZ) parents (B6; 129S-Oxt(tm1Wsy)/J mice) were bred in our animal facility. Male OXT-KO, WT and HZ littermates were used for the study. Solid gastric emptying was measured following acute restraint stress (for 90 min) or repeated restraint stress (for five consecutive days). Expression of CRF mRNA in the paraventricular nucleus (PVN) was measured by real-time reverse transcriptase-polymerase chain reaction. There were no significant differences of gastric emptying in WT (68.4 ± 4.1%, n = 6), HZ (71.8 ± 3.1%, n = 6) and OXT-KO (70.6 ± 3.1%, n = 6) mice in nonstressed conditions. Acute stress significantly delayed gastric emptying in OXT-KO mice (33.10 ± 2.5%, n = 6) WT (39.1 ± 1.1%, n = 6) and HZ mice (35.8 ± 1.2%, n = 6). Following repeated restraint stress loading, gastric emptying was significantly restored in WT (68.3 ± 4.5%, n = 6) and HZ mice (63.1 ± 2.6%, n = 6). By contrast, gastric emptying was still delayed in OXT-KO mice (34.7 ± 1.3%, n = 6) following repeated restraint stress. The increase in CRF mRNA expression at the PVN was much pronounced in OXT-KO mice compared to WT or HZ mice following repeated restraint stress. These findings suggest that central oxytocin plays a pivotal role in mediating the adaptation mechanism following repeated restraint stress in mice. PMID:20969650

  16. Arginase Inhibition in Airways from Normal and Nitric Oxide Synthase 2-Knockout Mice Exposed to Ovalbumin

    PubMed Central

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; O’Roark, Erin M.; Kenyon, Nicholas J.; Last, Jerold A.

    2011-01-01

    Arginase1 and nitric oxide synthase2 (NOS2) utilize L-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) significantly increased total L-arginine content in the airway compartment. We hypothesized that such an increase in L-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyper-reactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2−/−) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyper-reactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyper-reactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2−/− mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyper-reactivity in all treatment groups. NOS2−/− mice had a significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2−/− mice. We conclude that L-arginine metabolism plays an important role in the development of airway hyper-reactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be

  17. Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

    PubMed

    Bratt, Jennifer M; Franzi, Lisa M; Linderholm, Angela L; O'Roark, Erin M; Kenyon, Nicholas J; Last, Jerold A

    2010-01-01

    Arginase1 and nitric oxide synthase2 (NOS2) utilize l-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N(omega)-hydroxy-nor-l-arginine (nor-NOHA) significantly increased total l-arginine content in the airway compartment. We hypothesized that such an increase in l-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that l-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2, which

  18. β-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in β2-Adrenoceptor Knockout Mice

    PubMed Central

    Propping, Stefan; Lorenz, Kristina; Michel, Martin C.; Wirth, Manfred P.; Ravens, Ursula

    2016-01-01

    Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological

  19. Nox2 Knockout Delays Infarct Progression and Increases Vascular Recovery through Angiogenesis in Mice following Ischaemic Stroke with Reperfusion

    PubMed Central

    McCann, Sarah K.; Dusting, Gregory J.; Roulston, Carli L.

    2014-01-01

    Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation. PMID:25375101

  20. Elucidating the mechanisms of double ionization using intense half-cycle, single-cycle, and double half-cycle pulses

    SciTech Connect

    Kamta, G. Lagmago; Starace, Anthony F.

    2003-10-01

    We investigate the interaction of a two-active electron system (Li{sup -}) with intense single-cycle and double half-cycle pulses. The 'intensity' and 'frequency' considered correspond to the 'multiphoton above-barrier regime'. For the single-cycle pulse (SCP), the electric field changes sign once, allowing electron wave packets created during the first half cycle to recollide with the parent ion when driven back by the field. For the double half-cycle pulse (DHP), however, the electric field does not change sign, and electron wave packets created during the first half cycle are not driven back to the parent ion. We find that both single and double ionization are significantly larger for the SCP than for the DHP, thereby elucidating the role of the rescattering mechanism. On the other hand, doubly ionized electrons produced by a half-cycle pulse and a DHP are found to have angular distributions in which one electron is ejected in the direction of the pulse field, and the other in the opposite direction. This clear signature of electron correlations suggests that 'shake-off', 'knockout', and, possibly, 'multiphoton-sharing' processes are alternative contributing mechanisms for double ionization in this regime.

  1. Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates.

    PubMed

    Mienda, Bashir Sajo; Shamsir, Mohd Shahir; Illias, Rosli Md

    2016-04-01

    The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35gl(-1) and 1.40gl(-1) from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals. PMID:26878126

  2. Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice.

    PubMed

    Westbrook, Reyhan; Bonkowski, Michael S; Arum, Oge; Strader, April D; Bartke, Andrzej

    2014-01-01

    Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice. PMID:23833202

  3. The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

    PubMed Central

    Xiong, Qiuhong; Ünal, Can; Matthias, Jan; Steinert, Michael; Eichinger, Ludwig

    2015-01-01

    Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16− and ATG9−/16− cells and compare them to the previously reported ATG9− mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9− and ATG16− cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9−/16− double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9−/16− cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9−/16− cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes. PMID:25878144

  4. Effect of Global ATGL Knockout on Murine Fasting Glucose Kinetics

    PubMed Central

    Coelho, Margarida; Nunes, Patricia; Mendes, Vera M.; Manadas, Bruno; Heerschap, Arend; Jones, John G.

    2015-01-01

    Mice deficient in adipose triglyceride lipase (ATGL−/−) present elevated ectopic lipid levels but are paradoxically glucose-tolerant. Measurement of endogenous glucose production (EGP) and Cori cycle activity provide insights into the maintenance of glycemic control in these animals. These parameters were determined in 7 wild-type (ATGL+/−) and 6 ATGL−/− mice by a primed-infusion of [U-13C6]glucose followed by LC-MS/MS targeted mass-isotopomer analysis of blood glucose. EGP was quantified by isotope dilution of [U-13C6]glucose while Cori cycling was estimated by analysis of glucose triose 13C-isotopomers. Fasting plasma free fatty-acids were significantly lower in ATGL−/− versus control mice (0.43 ± 0.05 mM versus 0.73 ± 0.11 mM, P < 0.05). Six-hour fasting EGP rates were identical for both ATGL−/− and control mice (79 ± 11 versus 71 ± 7 μmol/kg/min, resp.). Peripheral glucose metabolism was dominated by Cori cycling (80 ± 2% and 82 ± 7% of glucose disposal for ATGL−/− and control mice, resp.) indicating that peripheral glucose oxidation was not significantly upregulated in ATGL−/− mice under these conditions. The glucose 13C-isotopomer distributions in both ATGL−/− and control mice were consistent with extensive hepatic pyruvate recycling. This suggests that gluconeogenic outflow from the Krebs cycle was also well compensated in ATGL−/− mice. PMID:26236747

  5. K Basins floor sludge retrieval system knockout pot basket fuel burn accident

    SciTech Connect

    HUNT, J.W.

    1998-11-11

    The K Basins Sludge Retrieval System Preliminary Hazard Analysis Report (HNF-2676) identified and categorized a series of potential accidents associated with K Basins Sludge Retrieval System design and operation. The fuel burn accident was of concern with respect to the potential release of contamination resulting from a runaway chemical reaction of the uranium fuel in a knockout pot basket suspended in the air. The unmitigated radiological dose to an offsite receptor from this fuel burn accident is calculated to be much less than the offsite risk evaluation guidelines for anticipated events. However, because of potential radiation exposure to the facility worker, this accident is precluded with a safety significant lifting device that will prevent the monorail hoist from lifting the knockout pot basket out of the K Basin water pool.

  6. Efficient gene knockout in goats using CRISPR/Cas9 system.

    PubMed

    Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A; Chen, Chuangfu

    2014-01-01

    The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications. PMID:25188313

  7. Podocyte-specific knockout of myosin 1e disrupts glomerular filtration.

    PubMed

    Chase, Sharon E; Encina, Christina V; Stolzenburg, Lindsay R; Tatum, Arthur H; Holzman, Lawrence B; Krendel, Mira

    2012-10-01

    Myosin 1e (myo1e) is an actin-dependent molecular motor that plays an important role in kidney functions. Complete knockout of myo1e in mice and Myo1E mutations in humans are associated with nephrotic syndrome and focal segmental glomerulosclerosis. In this paper, we tested the hypothesis that myo1e is necessary for normal functions of glomerular visceral epithelial cells (podocytes) using podocyte-targeted knockout of myo1e. Myo1e was selectively knocked out in podocytes using Cre-mediated recombination controlled by the podocin promoter. Myo1e loss from podocytes resulted in proteinuria, podocyte foot process effacement, and glomerular basement membrane disorganization. Our findings indicate that myo1e expression in podocytes is necessary for normal glomerular filtration and that podocyte defects are likely to represent the primary pathway leading to glomerular disease associated with Myo1E mutations. PMID:22811491

  8. Germ cell differentiation and synaptonemal complex formation are disrupted in CPEB knockout mice.

    PubMed

    Tay, J; Richter, J D

    2001-08-01

    CPEB is a sequence-specific RNA binding protein that regulates translation during vertebrate oocyte maturation. Adult female CPEB knockout mice contained vestigial ovaries that were devoid of oocytes; ovaries from mid-gestation embryos contained oocytes that were arrested at the pachytene stage. Male CPEB null mice also contained germ cells arrested at pachytene. The germ cells from the knockout mice harbored fragmented chromatin, suggesting a possible defect in homologous chromosome adhesion or synapsis. Two CPE-containing synaptonemal complex protein mRNAs, which interact with CPEB in vitro and in vivo, contained shortened poly(A) tails and mostly failed to sediment with polysomes in the null mice. Synaptonemal complexes were not detected in these animals. CPEB therefore controls germ cell differentiation by regulating the formation of the synaptonemal complex. PMID:11702780

  9. Knockout reactions from p-shell nuclei : tests of ab initio structure models.

    SciTech Connect

    Grinyer, G. F.; Bazin, D.; Gade, A.; Tostevin, J. A.; Adrich, P.; Bowen, M. D.; Brown, B. A.; Campbell, C. M.; Cook, J. M.; Glasmacher, T.; McDaniel, S.; Navratil, P.; Obertelli, A.; Quaglioni, S.; Siwek, K.; Terry, J. R.; Weisshaar, D.; Wiringa, R. B.

    2011-04-22

    Absolute cross sections have been determined following single neutron knockout reactions from {sup 10}Be and {sup 10}C at intermediate energy. Nucleon density distributions and bound-state wave function overlaps obtained from both variational Monte Carlo (VMC) and no core shell model (NCSM) ab initio calculations have been incorporated into the theoretical description of knockout reactions. Comparison to experimental cross sections demonstrates that the VMC approach, with the inclusion of 3-body forces, provides the best overall agreement while the NCSM and conventional shell-model calculations both overpredict the cross sections by 20% to 30% for {sup 10}Be and by 40% to 50% for {sup 10}C, respectively. This study gains new insight into the importance of 3-body forces and continuum effects in light nuclei and provides a sensitive technique to assess the accuracy of ab initio calculations for describing these effects.

  10. Knockout Reactions from p-Shell Nuclei: Tests of Ab Initio Structure Models

    SciTech Connect

    Grinyer, G. F.; Bazin, D.; Adrich, P.; Obertelli, A.; Weisshaar, D.; Gade, A.; Bowen, M. D.; Brown, B. A.; Campbell, C. M.; Cook, J. M.; Glasmacher, T.; McDaniel, S.; Siwek, K.; Terry, J. R.; Tostevin, J. A.; Navratil, P.; Quaglioni, S.; Wiringa, R. B.

    2011-04-22

    Absolute cross sections have been determined following single neutron knockout reactions from {sup 10}Be and {sup 10}C at intermediate energy. Nucleon density distributions and bound-state wave function overlaps obtained from both variational Monte Carlo (VMC) and no core shell model (NCSM) ab initio calculations have been incorporated into the theoretical description of knockout reactions. Comparison to experimental cross sections demonstrates that the VMC approach, with the inclusion of 3-body forces, provides the best overall agreement while the NCSM and conventional shell-model calculations both overpredict the cross sections by 20% to 30% for {sup 10}Be and by 40% to 50% for {sup 10}C, respectively. This study gains new insight into the importance of 3-body forces and continuum effects in light nuclei and provides a sensitive technique to assess the accuracy of ab initio calculations for describing these effects.

  11. Altered behavioral development in Nrf2 knockout mice following early postnatal exposure to valproic acid

    PubMed Central

    Furnari, Melody A.; Saw, Constance Lay-Lay; Kong, Ah-Ng; Wagner, George C

    2015-01-01

    Early exposure to valproic acid results in autism-like neural and behavioral deficits in humans and other animals through oxidative stress-induced neural damage. In the present study, valproic acid was administered to genetically altered mice lacking the Nrf2 (nuclear factor-erythroid 2 related factor 2) gene on postnatal day 14 (P14). Nrf2 is a transcription factor that induces genes that protect against oxidative stress. It was found that valproic acid-treated Nrf2 knockout mice were less active in open field activity chambers, less successful on the rotorod, and had deficits in learning and memory in the Morris water maze compared to the valproic acid-treated wild type mice. Given these results, it appears that Nrf2 knockout mice were more sensitive to the neural damage caused by valproic acid administered during early development. PMID:25454122

  12. Cnga2 Knockout Mice Display Alzheimer's-Like Behavior Abnormities and Pathological Changes.

    PubMed

    Xie, Ao-Ji; Liu, En-Jie; Huang, He-Zhou; Hu, Yu; Li, Ke; Lu, Youming; Wang, Jian-Zhi; Zhu, Ling-Qiang

    2016-09-01

    Olfactory dysfunction is recognized as a potential risk factor for Alzheimer's disease (AD). We have reported previously that olfactory deprivation by olfactory bulbectomy (OBX) induced Alzheimer's-like pathological changes and behavioral abnormalities. However, the acute OBX model undergoes surgical-induced brain parenchyma loss and unexpected massive hemorrhage so that it cannot fully mimic the progressive olfactory loss and neurodegeneration in AD. Here, we employed the mice loss of cyclic nucleotide-gated channel alpha 2 (Cnga2) which is critical for olfactory sensory transduction, to investigate the role of olfactory dysfunction in AD pathological process. We found that impaired learning and memory abilities, loss of dendrite spines, as well as decrement of synaptic proteins were displayed in Cnga2 knockout mice. Moreover, Aβ overproduction, tau hyperphosphorylation, and somatodendritic translocation were also found in Cnga2 knockout mice. Our findings suggest that progressive olfactory loss leads to Alzheimer's-like behavior abnormities and pathological changes. PMID:26377105

  13. Protein Production with a Pichia pastoris OCH1 Knockout Strain in Fed-Batch Mode.

    PubMed

    Gmeiner, Christoph; Spadiut, Oliver

    2015-01-01

    The methylotrophic yeast Pichia pastoris is a widely used host organism for recombinant protein production in biotechnology and pharmaceutical industry. However, if the target product describes a glycoprotein, an α-1,6-mannosyltransferase located in the Golgi apparatus of P. pastoris, called OCH1, triggers hypermannosylation of the recombinant protein which significantly impedes following unit operations and hampers biopharmaceutical product applications. A knockout of the och1 gene allows the production of less-glycosylated proteins-however, morphology and physiology of P. pastoris also change, complicating the upstream process. Here, we describe a controlled and efficient bioprocess based on the specific substrate uptake rate (q s) for a recombinant P. pastoris OCH1 knockout strain expressing a peroxidase as model protein. PMID:26082217

  14. CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity.

    PubMed

    Cao, Li; Qin, Xing; Peterson, Matthew R; Haller, Samantha E; Wilson, Kayla A; Hu, Nan; Lin, Xin; Nair, Sreejayan; Ren, Jun; He, Guanglong

    2016-03-01

    Obesity is associated with chronic inflammation which plays a critical role in the development of cardiovascular dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) in macrophages regulates innate immune responses via activation of pro-inflammatory cytokines, we hypothesize that CARD9 mediates the pro-inflammatory signaling associated with obesity en route to myocardial dysfunction. C57BL/6 wild-type (WT) and CARD9(-/-) mice were fed normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5months. At the end of 5-month HFD feeding, cardiac function was evaluated using echocardiography. Cardiomyocytes were isolated and contractile properties were measured. Immunofluorescence was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from isolated macrophages were collected to measure the concentrations of pro-inflammatory cytokines using ELISA kits. Western immunoblotting analyses were performed on heart tissue homogenates and isolated macrophages to explore the underlying signaling mechanism(s). CARD9 knockout alleviated HFD-induced insulin resistance and glucose intolerance, prevented myocardial dysfunction with preserved cardiac fractional shortening and cardiomyocyte contractile properties. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart with reduced myocardium-, plasma-, and macrophage-derived cytokines including IL-6, IL-1β and TNFα. Finally, CARD9 knockout abrogated the increase of p38 MAPK phosphorylation, the decrease of LC3BII/LC3BI ratio and the up-regulation of p62 expression in the heart induced by HFD feeding and restored cardiac autophagy signaling. In conclusion, CARD9 knockout ameliorates myocardial dysfunction associated with HFD-induced obesity, potentially through reduction of macrophage infiltration, suppression of p38 MAPK phosphorylation, and preservation of autophagy in the heart. PMID

  15. Transgenic knockout mice with exclusively human sickle hemoglobinand sickle cell disease

    SciTech Connect

    Paszty, C.; Brion, C.; Manci, E.; Witkowska, E.; Stevens, M.; Narla, M.; Rubin, E.

    1997-06-13

    To create mice expressing exclusively human sicklehemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, andbeta[S]-globin were generated and bred with knockout mice that haddeletions of the murine alpha- and beta-globin genes. These sickle cellmice have the major features (irreversibly sickled red cells, anemia,multiorgan pathology) found in humans with sickle cell disease and, assuch, represent a useful in vivo system to accelerate the development ofimproved therapies for this common genetic disease.

  16. Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.

    PubMed

    Shen, Baoming; Xiao, Jiling; Dai, Liangying; Huang, Yonghong; Mao, Zhenchuan; Lin, Runmao; Yao, Yurong; Xie, Bingyan

    2015-01-01

    The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150μgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient

  17. Kv4.2 Knockout Mice Have Hippocampal-Dependent Learning and Memory Deficits

    ERIC Educational Resources Information Center

    Lugo, Joaquin N.; Brewster, Amy L.; Spencer, Corinne M.; Anderson, Anne E.

    2012-01-01

    Kv4.2 channels contribute to the transient, outward K[superscript +] current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit…

  18. Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency.

    PubMed

    Daude, Nathalie; Wohlgemuth, Serene; Brown, Rebecca; Pitstick, Rose; Gapeshina, Hristina; Yang, Jing; Carlson, George A; Westaway, David

    2012-06-01

    The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP(C)). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP(C)-like function to maintain the viability of Prnp(0/0) mice lacking the PrP(C) protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a "C1" C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, "N1," which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg1 gene transcription unit that overlaps the Sprn 3' UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn(0/0) mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp(0/0) embryos using lentiviruses targeted against the Sprn 3' UTR, we established that double-knockout mice deficient in both Sho and PrP(C) are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrP(C) and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP(C), we infer that Sho's activity will prove germane to the maintenance of neuronal viability in postnatal life. PMID:22619325

  19. Topological Quantum Double

    NASA Astrophysics Data System (ADS)

    Bonneau, Philippe

    Following a preceding paper showing how the introduction of a t.v.s. topology on quantum groups led to a remarkable unification and rigidification of the different definitions, we adapt here, in the same way, the definition of quantum double. This topological double is dualizable and reflexive (even for infinite dimensional algebras). In a simple case we show, considering the double as the "zero class" of an extension theory, the uniqueness of the double structure as a quasi-Hopf algebra. A la suite d'un précédent article montrant comment l'introduction d'une topologie d'e.v.t. sur les groupes quantiques permet une unification et une rigidification remarquables des différentes définitions, on adapte ici de la même manière la définition du double quantique. Ce double topologique est alors dualisable et reflexif (même pour des algèbres de dimension infinie). Dans un cas simple on montre, en considérant le double comme la "classe zéro" d'une théorie d'extensions, l'unicité de cette structure comme algèbre quasi-Hopf.

  20. Emerging double helical nanostructures

    NASA Astrophysics Data System (ADS)

    Zhao, Meng-Qiang; Zhang, Qiang; Tian, Gui-Li; Wei, Fei

    2014-07-01

    As one of the most important and land-mark structures found in nature, a double helix consists of two congruent single helices with the same axis or a translation along the axis. This double helical structure renders the deoxyribonucleic acid (DNA) the crucial biomolecule in evolution and metabolism. DNA-like double helical nanostructures are probably the most fantastic yet ubiquitous geometry at the nanoscale level, which are expected to exhibit exceptional and even rather different properties due to the unique organization of the two single helices and their synergistic effect. The organization of nanomaterials into double helical structures is an emerging hot topic for nanomaterials science due to their promising exceptional unique properties and applications. This review focuses on the state-of-the-art research progress for the fabrication of double-helical nanostructures based on `bottom-up' and `top-down' strategies. The relevant nanoscale, mesoscale, and macroscopic scale fabrication methods, as well as the properties of the double helical nanostructures are included. Critical perspectives are devoted to the synthesis principles and potential applications in this emerging research area. A multidisciplinary approach from the scope of nanoscience, physics, chemistry, materials, engineering, and other application areas is still required to the well-controlled and large-scale synthesis, mechanism, property, and application exploration of double helical nanostructures.

  1. Neutron Knockout to Probe 3N Forces in the Ca Isotopes

    NASA Astrophysics Data System (ADS)

    Crawford, Heather; NSCL Experiment e12029 Collaboration

    2013-10-01

    Recent calculations by Holt et al. have suggested that the inclusion of 3N forces to describe the structure of neutron-rich Ca isotopes provides a more realistic description of the nuclear structure. Mass measurements have confirmed the importance of including 3N forces compared to NN-only interactions, but cannot discriminate between the predictions of phenomenological interactions and the NN+3N microscopic calculations. Neutron knockout along the Z = 20 isotopes provides an opportunity to test the results of NN+3N calculations against phenomenological interactions. The calculations of Holt et al. predict a fragmentation of the 1f7 / 2 neutron strength from the first 7/2- state in 49Ca into higher lying states, in contrast to the predictions of both GXPF1 and KB3G which concentrate the strength in the lowest lying 7/2- state. Differences are also observed in the summed f7 / 2 strength to bound nuclear states in both 50Ca and 49Ca neutron knockout. We will report on a systematic study of one-neutron knockout along the Ca isotopes using GRETINA+S800. Comparison of experimental spectroscopic factors to calculations will provide insight into the role of 3N forces in describing the Z = 20 isotopes. With collaborators from LBNL, NSCL/MSU, ANL, Central Michigan University, TRIUMF/UBC and TU-Darmstadt.

  2. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout.

    PubMed

    Banati, Richard B; Middleton, Ryan J; Chan, Ronald; Hatty, Claire R; Kam, Winnie Wai-Ying; Quin, Candice; Graeber, Manuel B; Parmar, Arvind; Zahra, David; Callaghan, Paul; Fok, Sandra; Howell, Nicholas R; Gregoire, Marie; Szabo, Alexander; Pham, Tien; Davis, Emma; Liu, Guo-Jun

    2014-01-01

    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs. PMID:25406832

  3. Myo5b knockout mice as a model of microvillus inclusion disease

    PubMed Central

    Cartón-García, Fernando; Overeem, Arend W.; Nieto, Rocio; Bazzocco, Sarah; Dopeso, Higinio; Macaya, Irati; Bilic, Josipa; Landolfi, Stefania; Hernandez-Losa, Javier; Schwartz, Simo; Ramon y Cajal, Santiago; van Ijzendoorn, Sven C. D.; Arango, Diego

    2015-01-01

    Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and development of novel therapeutic approaches is hampered by the lack of animal models. In this study we describe the phenotype of a novel mouse model with targeted inactivation of Myo5b. Myo5b knockout mice show perinatal mortality, diarrhea and the characteristic mislocalization of apical and basolateral plasma membrane markers in enterocytes. Moreover, in transmission electron preparations, we observed microvillus atrophy and the presence of microvillus inclusion bodies. Importantly, Myo5b knockout embryos at day 20 of gestation already display all these structural defects, indicating that they are tissue autonomous rather than secondary to environmental cues, such as the long-term absence of nutrients in the intestine. Myo5b knockout mice closely resemble the phenotype of MVID patients and constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches. PMID:26201991

  4. Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.

    PubMed

    Boshra, Hani; Cao, Jingxin; Babiuk, Shawn

    2016-01-01

    The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses. PMID:26458835

  5. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout

    PubMed Central

    Banati, Richard B.; Middleton, Ryan J.; Chan, Ronald; Hatty, Claire R.; Wai-Ying Kam, Winnie; Quin, Candice; Graeber, Manuel B.; Parmar, Arvind; Zahra, David; Callaghan, Paul; Fok, Sandra; Howell, Nicholas R.; Gregoire, Marie; Szabo, Alexander; Pham, Tien; Davis, Emma; Liu, Guo-Jun

    2014-01-01

    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer’s disease to anxiety. Here we show that global C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from GuwiyangWurraTSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of GuwiyangWurraTSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs. PMID:25406832

  6. The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats

    PubMed Central

    Zhuang, Zhao-Meng; Wang, Xiao-Teng; Zhang, Lu; Tao, Li-Yuan; Lv, Bin

    2015-01-01

    Objective: To observe the changes of intestinal inflammation on PDIA3 gene knockout IBS rats and its effect on immune function. Methods: 36 SD rats were randomly divided into four groups: the control group (n = 8); IBS- empty virus group (IBS-GFP, which); IBS-PDIA3 knockout group (n = 12); IBS- the control group (n = 12). After modeling, colon and ileocecal tissue pathology in each group were observed separately. Changes of immune and inflammatory markers were measured. At the same time, ultrastructural changes in each group were observed by electron microscopy. Results: Compared with the IBS control group, inflammation was reduced significantly in IBS-PDIA3 knockout group. IgE, IL-4 and IL-9 and the level of intestinal trypsin type were decreased significantly. Furthermore, mast cell degranulation and PAR 2 receptor reduced significantly. Conclusion: PDIA3 may play an important role in the development of IBS by mediating through immune responses of mucosal abnormalities. However, the mechanism needs to be confirmed in further study. PMID:26221224

  7. Identification of Novel Knockout Targets for Improving Terpenoids Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8–10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids. PMID:25386654

  8. Higher spin double field theory: a proposal

    NASA Astrophysics Data System (ADS)

    Bekaert, Xavier; Park, Jeong-Hyuck

    2016-07-01

    We construct a double field theory coupled to the fields present in Vasiliev's equations. Employing the "semi-covariant" differential geometry, we spell a functional in which each term is completely covariant with respect to O(4, 4) T-duality, doubled diffeomorphisms, Spin(1, 3) local Lorentz symmetry and, separately, HS(4) higher spin gauge symmetry. We identify a minimal set of BPS-like conditions whose solutions automatically satisfy the full Euler-Lagrange equations. As such a solution, we derive a linear dilaton vacuum. With extra algebraic constraints further supplemented, the BPS-like conditions reduce to the bosonic Vasiliev equations.

  9. Revisiting double Dirac delta potential

    NASA Astrophysics Data System (ADS)

    Ahmed, Zafar; Kumar, Sachin; Sharma, Mayank; Sharma, Vibhu

    2016-07-01

    We study a general double Dirac delta potential to show that this is the simplest yet still versatile solvable potential to introduce double wells, avoided crossings, resonances and perfect transmission (T = 1). Perfect transmission energies turn out to be the critical property of symmetric and anti-symmetric cases wherein these discrete energies are found to correspond to the eigenvalues of a Dirac delta potential placed symmetrically between two rigid walls. For well(s) or barrier(s), perfect transmission (or zero reflectivity, R(E)) at energy E=0 is non-intuitive. However, this has been found earlier and called the ‘threshold anomaly’. Here we show that it is a critical phenomenon and we can have 0≤slant R(0)\\lt 1 when the parameters of the double delta potential satisfy an interesting condition. We also invoke a zero-energy and zero curvature eigenstate (\\psi (x)={Ax}+B) of the delta well between two symmetric rigid walls for R(0)=0. We resolve that the resonant energies and the perfect transmission energies are different and they arise differently.

  10. Phosphorylation of 4EBP by oral leucine administration was suppressed in the skeletal muscle of PGC-1α knockout mice.

    PubMed

    Yoshimura, Ryoji; Minami, Kimiko; Matsuda, Junichiro; Sawada, Naoki; Miura, Shinji; Kamei, Yasutomi

    2016-01-01

    Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis. PMID:26745679

  11. Double Degenerate Binary Systems

    SciTech Connect

    Yakut, K.

    2011-09-21

    In this study, angular momentum loss via gravitational radiation in double degenerate binary (DDB)systems (NS + NS, NS + WD, WD + WD, and AM CVn) is studied. Energy loss by gravitational waves has been estimated for each type of systems.

  12. Double aortic arch

    MedlinePlus

    ... double aortic arch may press on the windpipe (trachea) and esophagus, leading to trouble breathing and swallowing. ... to relieve pressure on the esophagus and windpipe (trachea). The surgeon ties off the smaller branch and ...

  13. Tissue-specific knockouts of ACAT2 reveal that intestinal depletion is sufficient to prevent diet-induced cholesterol accumulation in the liver and blood[S

    PubMed Central

    Zhang, Jun; Kelley, Kathryn L.; Marshall, Stephanie M.; Davis, Matthew A.; Wilson, Martha D.; Sawyer, Janet K.; Farese, Robert V.; Brown, J. Mark; Rudel, Lawrence L.

    2012-01-01

    Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2L−/L−) and intestine-specific (ACAT2SI−/SI−) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2SI−/SI− mice, in contrast to ACAT2L−/L− mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2L−/L− and ACAT2SI−/SI− mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection. PMID:22460046

  14. Efficiency of primary saliva secretion: an analysis of parameter dependence in dynamic single-cell and acinus models, with application to aquaporin knockout studies

    PubMed Central

    Maclaren, Oliver J.; Sneyd, James; Crampin, Edmund J.

    2012-01-01

    Secretion from the salivary glands is driven by osmosis following the establishment of osmotic gradients between the lumen, the cell and the interstitium by active ion transport. We consider a dynamic model of osmotically-driven primary saliva secretion, and use singular perturbation approaches and scaling assumptions to reduce the model. Our analysis shows that isosmotic secretion is the most efficient secretion regime, and that this holds for single isolated cells and for multiple cells assembled into an acinus. For typical parameter variations, we rule out any significant synergistic effect on total water secretion of an acinar arrangement of cells about a single shared lumen. Conditions for the attainment of isosmotic secretion are considered, and we derive an expression for how the concentration gradient between the interstitium and the lumen scales with water and chloride transport parameters. Aquaporin knockout studies are interpreted in the context of our analysis and further investigated using simulations of transport efficiency with different membrane water permeabilities. We conclude that recent claims that aquaporin knockout studies can be interpreted as evidence against a simple osmotic mechanism are not supported by our work. Many of the results that we obtain are independent of specific transporter details, and our analysis can be easily extended to apply to models that use other proposed ionic mechanisms of saliva secretion. PMID:22258315