Elastic medium confined in a column versus the Janssen experiment

We compute the stresses in an elastic medium confined in a vertical column, when the material is at the Coulomb threshold everywhere at the walls. Simulations are performed in 2 dimensions using a spring lattice, and in 3 dimensions, using Finite Element Method. The results are compared to the Janssen model and to experimental results for a granular material. The necessity to consider elastic anisotropy to render qualitatively the experimental findings is discussed

No self-similar aggregates with sedimentation

Two-dimensional cluster-cluster aggregation is studied when clusters move both diffusively and sediment with a size dependent velocity. Sedimentation breaks the rotational symmetry and the ensuing clusters are not self-similar fractals: the mean cluster width perpendicular to the field direction grows faster than the height. The mean width exhibits power-law scaling with respect to the cluster size, ~ s^{l_x}, l_x = 0.61 +- 0.01, but the mean height does not. The clusters tend to become elongated in the sedimentation direction and the ratio of the single particle sedimentation velocity to single particle diffusivity controls the degree of orientation. These results are obtained using a simulation method, which becomes the more efficient the larger the moving clusters are.Comment: 10 pages, 10 figure

The Gp1ba-Cre transgenic mouse::A new model to delineate platelet and leukocyte functions

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved

Phase Transition in Perovskite Manganites with Orbital Degree of Freedom

Roles of orbital degree of freedom of Mn ions in phase transition as a function of temperature and hole concentration in perovskite manganites are studied. It is shown that the orbital order-disorder transition is of the first order in the wide region of hole concentration and the N$\rm \acute{e}$el temperature for the anisotropic spin ordering, such as the layer-type antiferromagnetic one, is lower than the orbital ordering temperature due to the anisotropy in the orbital space. The calculated results of the temperature dependence of the spin and orbital order parameters explain a variety of the experiments observed in manganites.Comment: 10 pages, 5 figure

Strain effect on electronic transport and ferromagnetic transition temperature in La0.9_{0.9}Sr0.1_{0.1}MnO3_{3} thin films

We report on a systematic study of strain effects on the transport properties and the ferromagnetic transition temperature $T_{c}$ of high-quality La$_{0.9}$Sr$_{0.1}$MnO$_{3}$ thin films epitaxially grown on (100) SrTiO$_{3}$ substrates. Both the magnetization and the resistivity are critically dependent on the film thickness. $T_{c}$ is enhanced with decreasing the film thickness due to the compressive stain produced by lattice mismatch. The resistivity above 165 K of the films with various thicknesses is consistent with small polaronic hopping conductivity. The polaronic formation energy $E_{P}$ is reduced with the decrease of film thickness. We found that the strain dependence of $T_{c}$ mainly results from the strain-induced electron-phonon coupling. The strain effect on $E_{P}$ is in good agreement with the theoretical predictions.Comment: 6 pages and 5 figures, accepted for publication in Phys. Rev.

Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice.

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor

Minimal regulation of platelet activity by PECAM-1

PECAM-1 is a member of the superfamily of immunoglobulins (Ig) and is expressed on platelets at moderate level. PECAM-1 has been reported to have contrasting effects on platelet activation by the collagen receptor GPVI and the integrin, αIIbβ3, even though both receptors signal through Src-kinase regulation of PLCγ2. The present study compares the role of PECAM-1 on platelet activation by these two receptors and by the lectin receptor, CLEC-2, which also signals via PLCγ2. Studies using PECAM-1 knockout-mice and cross-linking of PECAM-1 using specific antibodies demonstrated a minor inhibitory role on platelet responses to the above three receptors and also under some conditions to the G-protein agonist thrombin. The degree of inhibition was considerably less than that produced by PGI2, which elevates cAMP. There was no significant difference in thrombus formation on collagen in PECAM-1-/- platelets relative to litter-matched controls. The very weak inhibitory effect of PECAM-1 on platelet activation relative to that of PGI2 indicate that the Ig-receptor is not a major regulator of platelet activation. PECAM-1 has been reported to have contrasting effects on platelet activation. The present study demonstrates a very mild or negligible effect on platelet activation in response to stimulation by a variety of agonists, thereby questioning the physiological role of the immunoglobulin receptor as a major regulator of platelet activation

A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

[EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. Results: Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA con¿ struction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. Conclusions: The functionality and the efficiency of gRNA¿Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA¿Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8. doi: 10.1016/j.cell.2013.04.025 .Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv. 2015;33(1):41–52. doi: 10.1016/j.biotechadv.2014.12.006 .Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol. 2015;32:76–84. doi: 10.1016/j.copbio.2014.11.007 .Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013;31(8):686–8. doi: 10.1038/nbt.2650 .Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol. 2015;87(1–2):99–110. doi: 10.1007/s11103-014-0263-0 .Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 2014;79(2):348–59. doi: 10.1111/tpj.12554 .Schiml S, Fauser F, Puchta H. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 2014;80(6):1139–50. doi: 10.1111/tpj.12704 .Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J. 2015;13(4):578–89. doi: 10.1111/pbi.12284 .Beerli RR, Barbas CF 3rd. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol. 2002;20(2):135–41. doi: 10.1038/nbt0202-135 .Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333(6051):1843–6. doi: 10.1126/science.1204094 .Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol. 2014;10:763. doi: 10.15252/msb.20145735 .Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J. Progress in plant protoplast research. Planta. 2013. doi: 10.1007/s00425-013-1936-7 .Mikami M, Toki S, Endo M. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol. 2015. doi: 10.1007/s11103-015-0342-x .Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol. 2015. doi: 10.1111/nph.13532 .Liu W, Stewart CN Jr. Plant synthetic biology. Trends Plant Sci. 2015;20(5):309–17. doi: 10.1016/j.tplants.2015.02.004 .Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013;162(3):1618–31. doi: 10.1104/pp.113.217661 .Vazquez-Vilar M, Sarrion-Perdigones A, Ziarsolo P, Blanca J, Granell A, Orzaez D. Software-assisted stacking of gene modules using GoldenBraid 2.0 DNA-assembly framework. Methods Mol Biol. 2015;1284:399–420. doi: 10.1007/978-1-4939-2444-8_20 .Duportet X, Wroblewska L, Guye P, Li Y, Eyquem J, Rieders J, et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. 2014;42(21):13440–51. doi: 10.1093/nar/gku1082 .Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, et al. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015;43(13):e88. doi: 10.1093/nar/gkv464 .Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553 .Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE. 2011;6(7):e21622. doi: 10.1371/journal.pone.0021622 .Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7(9):1494–6. doi: 10.1093/mp/ssu044 .Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. doi: 10.1126/science.1232033 .Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31(8):688–91. doi: 10.1038/nbt.2654 .Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37. doi: 10.1093/nar/gkt520 .Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA. 2015;112(11):3570–5. doi: 10.1073/pnas.1420294112 .Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE. 2011;6(2):e16765. doi: 10.1371/journal.pone.0016765 .Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep. 2014;4:5400. doi: 10.1038/srep05400 .Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015. doi: 10.1016/j.molp.2015.04.007 .Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015;169(2):971–85. doi: 10.1104/pp.15.00636 .Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31(8):691–3. doi: 10.1038/nbt.2655 .Upadhyay SK, Kumar J, Alok A, Tuli R. RNA-guided genome editing for target gene mutations in wheat. G3. 2013;3(12):2233–8. doi: 10.1534/g3.113.008847 .Senis E, Fatouros C, Grosse S, Wiedtke E, Niopek D, Mueller AK, et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J. 2014;9(11):1402–12. doi: 10.1002/biot.201400046 .Port F, Chen HM, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci USA. 2014;111(29):E2967–76. doi: 10.1073/pnas.1405500111 .Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014;14:327. doi: 10.1186/s12870-014-0327-y