An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene's function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generatio

Simulation of a Dripping Faucet

We present a simulation of a dripping faucet system. A new algorithm based on Lagrangian description is introduced. The shape of drop falling from a faucet obtained by the present algorithm agrees quite well with experimental observations. Long-term behavior of the simulation can reproduce period-one, period-two, intermittent and chaotic oscillations widely observed in experiments. Possible routes to chaos are discussed.Comment: 20 pages, 15 figures, J. Phys. Soc. Jpn. (in press

Proc. Nat. Acad. Sci. USA

Drosophila Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for the maintenance of stable transcription patterns of many developmental regulators, such as the homeotic genes. We have used ChIP-on-chip to compare the distribution of several PcG/TrxG proteins, as well as histone modifications in active and repressed genes across the two homeotic complexes ANT-C and BX-C. Our data indicate the colocalization of the Polycomb repressive complex 1 (PRC1) with Trx and the DNA binding protein Pleiohomeotic (Pho) at discrete sequence elements as well as significant chromatin assembly differences in active and inactive regions. Trx binds to the promoters of active genes and noncoding transcripts. Most strikingly, in the active state, Pho covers extended chromatin domains over many kilobases. This feature of Pho, observed on many polytene chromosome puffs, reflects a previously undescribed function. At the hsp70 gene, we demonstrate in mutants that Pho is required for transcriptional recovery after heat shock. Besides its presumptive function in recruiting PcG complexes to their site of action, our results now uncover that Pho plays an additional role in the repression of already induced genes

Dripping Faucet Dynamics Clarified by an Improved Mass-Spring Model

An improved mass-spring model for a dripping faucet is presented. The model is constructed based on the numerical results which we recently obtained from fluid dynamical calculations. Both the fluid dynamical calculations and the present mass-spring model exhibit a variety of complex behavior including transition to chaos in good agreement with experiments. Further, the mass-spring model reveals fundamental dynamics inherent in the dripping faucet system.Comment: 17 pages, 17 figure

Low thermal conductivity of the layered oxide (Na,Ca)Co_2O_4: Another example of a phonon glass and an electron crystal

The thermal conductivity of polycrystalline samples of (Na,Ca)Co_2O_4 is found to be unusually low, 20 mW/cmK at 280 K. On the assumption of the Wiedemann-Franz law, the lattice thermal conductivity is estimated to be 18 mW/cmK at 280 K, and it does not change appreciably with the substitution of Ca for Na. A quantitative analysis has revealed that the phonon mean free path is comparable with the lattice parameters, where the point-defect scattering plays an important role. Electronically the same samples show a metallic conduction down to 4.2 K, which strongly suggests that NaCo_2O_4 exhibits a glass-like poor thermal conduction together with a metal-like good electrical conduction. The present study further suggests that a strongly correlated system with layered structure can act as a material of a phonon glass and an electron crystal.Comment: 5 pages 3 figures, to be published in Phys. Rev.

Inertial- and Dissipation-Range Asymptotics in Fluid Turbulence

We propose and verify a wave-vector-space version of generalized extended self similarity and broaden its applicability to uncover intriguing, universal scaling in the far dissipation range by computing high-order (\leq 20\/) structure functions numerically for: (1) the three-dimensional, incompressible Navier Stokes equation (with and without hyperviscosity); and (2) the GOY shell model for turbulence. Also, in case (2), with Taylor-microscale Reynolds numbers 4 \times 10^{4} \leq Re_{\lambda} \leq 3 \times 10^{6}\/, we find that the inertial-range exponents (\zeta_{p}\/) of the order - p\/ structure functions do not approach their Kolmogorov value p/3\/ as Re_{\lambda}\/ increases.Comment: RevTeX file, with six postscript figures. epsf.tex macro is used for figure insertion. Packaged using the 'uufiles' utilit

The CD38/NAD/SIRTUIN1/EZH2 Axis Mitigates Cytotoxic CD8 T Cell Function and Identifies Patients with SLE Prone to Infections

Summary: Patients with systemic lupus erythematosus (SLE) suffer frequent infections that account for significant morbidity and mortality. T cell cytotoxic responses are decreased in patients with SLE, yet the responsible molecular events are largely unknown. We find an expanded CD8CD38high T cell subset in a subgroup of patients with increased rates of infections. CD8CD38high T cells from healthy subjects and patients with SLE display decreased cytotoxic capacity, degranulation, and expression of granzymes A and B and perforin. The key cytotoxicity-related transcription factors T-bet, RUNX3, and EOMES are decreased in CD8CD38high T cells. CD38 leads to increased acetylated EZH2 through inhibition of the deacetylase Sirtuin1. Acetylated EZH2 represses RUNX3 expression, whereas inhibition of EZH2 restores CD8 T cell cytotoxic responses. We propose that high levels of CD38 lead to decreased CD8 T cell-mediated cytotoxicity and increased propensity to infections in patients with SLE, a process that can be reversed pharmacologically. : Katsuyama et al. find that an expanded CD8CD38high T cell population in SLE patients is linked to infections. CD8CD38high T cells display decreased cytotoxic capacity by suppressing the expression of related molecules through an NAD+/Sirtuin1/EZH2 pathway. EZH2 inhibitors increase cytotoxicity offering a means to mitigate infection rates in SLE. Keywords: systemic lupus erythematosus, patients, CD8 T cell, CD38, cytotoxicity, infection, nicotinamide adenine dinucleotide, Sirtuin1, EZH

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides

Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis

Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.Portuguese Fundacao para Ciencia e Tecnologia (FCT) [PTDC/EXPL/MARBIO/0430/2013]; CCMAR FCT Plurianual financing [UID/Multi/04326/2013]; FCT [SFRH/BD/111226/2015, SFRH/BD/108842/2015, SFRH/BPD/89889/2012]; FCT-IF Starting Grant [IF/01274/2014]info:eu-repo/semantics/publishedVersio

SLAMF6 as a Regulator of Exhausted CD8+ T Cells in Cancer

The tumor microenvironment in leukemia and solid tumors induces a shift of activated CD8+ cytotoxic T cells to an exhausted state, characterized by loss of proliferative capacity and impaired immunologic synapse formation. Efficient strategies and targets need to be identified to overcome T-cell exhaustion and further improve overall responses in the clinic. Here, we took advantage of the Eμ-TCL1 chronic lymphocytic leukemia (CLL) and B16 melanoma mouse models to assess the role of the homophilic cell-surface receptor SLAMF6 as an immune-checkpoint regulator. The transfer of SLAMF6+ Eμ-TCL1 cells into SLAMF6−/− recipients, in contrast to wild-type (WT) recipients, significantly induced expansion of a PD-1+ subpopulation among CD3+CD44+CD8+ T cells, which had impaired cytotoxic functions. Conversely, administering anti-SLAMF6 significantly reduced the leukemic burden in Eμ-TCL1 recipient WT mice concomitantly with a loss of PD-1+CD3+CD44+CD8+ T cells with significantly increased effector functions. Anti-SLAMF6 significantly reduced leukemic burden in the peritoneal cavity, a niche where antibody-dependent cellular cytotoxicity (ADCC) is impaired, possibly through activation of CD8+ T cells. Targeting of SLAMF6 affected tumor growth not only in B cell–related leukemia and lymphomas but also in nonhematopoietic tumors such as B16 melanoma, where SLAMF6 is not expressed. In vitro exhausted CD8+ T cells showed increased degranulation when anti-human SLAMF6 was added in culture. Taken together, anti-SLAMF6 both effectively corrected CD8+ T-cell dysfunction and had a direct effect on tumor progression. The outcomes of our studies suggest that targeting SLAMF6 is a potential therapeutic strategy