Genetic biosensor enables in vivo glycosyltransferase screening

Glycosylation of natural products can alter their solubility and bioavailability, among other properties, which makes glycosyltransferases useful tools for increasing the production and/or generating novel compounds in microbial cell factories. However, the discovery and screening of new enzymes and engineered variants is often a low-throughput endeavor due to the need for over-expression and purification prior to in vitro experiments, which do not necessarily represent the in vivo activities of the enzyme. Therefore, a genetic biosensor controlling GFP expression was developed based on the flavonoid responsive transcriptional-repressor QdoR and expressed in E. coli. Due to the induced fluorescent response upon feeding the flavonoids Quercetin and Kaempferol, but not to their glucosides, the activity of UDP-dependent glycosyltransferases (UGTs) could be screened in vivo. Furthermore, a variant of QdoR was generated by directed evolution that showed greater dose-responsiveness and proved to allow greater discrimination of cellular populations and was thus more useful for in vivo UGT screening. The designed biosensor-based method will greatly increase the throughput of glycosyltransferase discovery and engineering. Please click Additional Files below to see the full abstract

The State of Education Savings Account Programs in the United States

Arguably the most strongly promoted approach by voucher advocates is a new form of government subsidy for private education, Education Savings Accounts (ESAs). Parents are provided a set sum which they can use for a variety of educational services including private school tuition and fees, online courses, extracurricular activities and private tutoring. Students enrolled in an ESA program are not allowed to concurrently attend a public school. This policy brief examines this emerging policy, considering similarities and differences with conventional voucher approaches, and examining the legal issues that the policy raises

A growth selection system for the directed evolution of Sucrose Synthases

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Small-molecule biosensors for high-throughput metabolic engineering

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. In this presentation we outline a versatile and high-throughput method to evolve and functionalize prokaryotic aTF ligand specificity and transfer functions in a eukaryote chassis, namely baker’s yeast Saccharomyces cerevisiae. From a single round of directed evolution of the aTF ligand-binding domain coupled with various toggled selection regimes, we robustly select aTF variants evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion of function from activation to repression. Importantly, by targeting only the ligand-binding domain, the evolved biosensors display DNA-binding affinities similar to parental aTFs and are functional when ported back into a non-native prokaryote chassis. The developed platform technology thus leverages aTF evolvability for the development of new biosensors with user-defined small-molecule specificities and transfer functions. Finally, the presentation will highlight examples on biosensor applications for high-throughput metabolic engineering. Please click Additional Files below to see the full abstract

Por que teoria é importante: Uma investigação das reformas contemporâneas do tempo de aprendizagem

This article explores the contemporary policy reform push to extend and expand learning time in schools. In light of the potential and continued prominence of learning time reforms in today’s national educational landscape, this article makes visible the ways in which theory matters for the near- and long-term success of equity-focused educational reforms. Using the recent enactment of learning time reforms in Colorado as an illustration, and the zone of mediation framework as a conceptual lens, this article demonstrates how such reforms are likely to be weakened and undermined without strong theoretical grounding.Este artículo examina políticas contemporáneas que enfatizan un cambio para extender y/o dispersar el tiempo de aprendizaje en las escuelas. Dado el potencial y relevancia actual de las reformas hacia el tiempo de aprendizaje en el campo general de educación, el propósito de este articulo es brevemente enaltecer las maneras en las cuales la teoría educativa son de suma importancia, a largo y corto plazo, para asegurar el éxito de dichas reformas educativas orientadas hacia la equidad. Utilizando el ejemplo de la implementación de estas reformas en Colorado, y el instrumento teorético de la zona me mediación, este artículo demuestra como reformas similares pueden ser disminuidos por una falta de un base de teoría fuerte.Este artigo examina o promover da reforma contemporânea das regras para ampliar e expandir o tempo de aprendizagem nas escolas. Considerando o potencial e o contínuo destaque da reforma do tempo de aprendizagem no ambiente da educação nacional atualmente, este artigo ilustra as maneiras em que teoria é importante para o sucesso imediato e futuro das reformas educacionais que se focalizam em equidade. Usando a recente promulgação das reformas do tempo de aprendizagem em Colorado como ilustração, e o sistema de zona de mediação como uma lente conceitual, o artigo demonstra como tais reformas serão provavelmente enfraquecidas sem uma forte base teórica

Acetylation of C/EBP alpha inhibits its granulopoietic function

CCAAT/enhancer-binding protein alpha (C/EBP alpha) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBP alpha at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBP alpha DNA-binding ability and modulates C/EBP alpha transcriptional activity. Acetylated C/EBP alpha is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)-mediated granulocytic differentiation of 32Dcl3 cells. C/EBP alpha mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBP alpha-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBP alpha and demonstrate the importance of C/EBP alpha acetylation in myeloid differentiation

Lysine acetyltransferase Tip60 is required for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.Cancer Research UK, Wellcome Trust, National Institutes of Health, Singapore state fundin

Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin− c-Kit+ Sca-1+ IL-7Rα−), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2−/− mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2−/− mice correlating with their higher fungal burden. Accordingly, emigration of Ly6Chigh monocytes and neutrophils to spleen was increased in TLR2−/− mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2−/− infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin− cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen

The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis

A recessive mutation named Justy was found that abolishes B lymphopoiesis but does not impair other major aspects of hematopoiesis. Transplantation experiments showed that homozygosity for Justy prevented hematopoietic progenitors from generating B cells but did not affect the ability of bone marrow stroma to support B lymphopoiesis. In bone marrow from mutant mice, common lymphoid progenitors and pre-pro–B cells appeared normal, but cells at subsequent stages of B lymphopoiesis were dramatically reduced in number. Under culture conditions that promoted B lymphopoiesis, mutant pre-pro–B cells remained alive and began expressing the B cell marker CD19 but failed to proliferate. In contrast, these cells were able to generate myeloid or T/NK precursors. Genetic and molecular analysis demonstrated that Justy is a point mutation within the Gon4-like (Gon4l) gene, which encodes a protein with homology to transcriptional regulators. This mutation was found to disrupt Gon4l pre-mRNA splicing and dramatically reduce expression of wild-type Gon4l RNA and protein. Consistent with a role for Gon4l in transcriptional regulation, the levels of RNA encoding C/EBPα and PU.1 were abnormally high in mutant B cell progenitors. Our findings indicate that the Gon4l protein is required for B lymphopoiesis and may function to regulate gene expression during this process