Temperature-induced ultraviolet difference absorption spectrometry for determination of enthalpy changes Binding of 4-methylumbelliferyl glycosides to four lectins

AbstractRaising the temperature in a single mixture of a lectin and a chromophoric glycoside allows determination of the binding enthalpy. This is made possible by continuously monitoring the displacement of the complex from its equilibrium concentration with a sensitive difference absorption spectrophotometer. The method is illustrated with the following lectins: concanavalin A, soybean agglutinin, peanut agglutinin and Erythrina cristagalli agglutinin. The ligands are 4-methylumbelliferyl glycosides. The binding enthalpies found range from −60 kJ · mol−1 for the Galß1 → 3GalNAc-ßglycoside and peanut agglutinin to −30 kJ · mol−1 for a monosaccharide glycoside and the other lectins

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results: We evaluated two suitable RNA isolation kits (theRNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5x dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells

PHF6 expression levels impact human hematopoietic stem cell differentiation

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in PHF6, encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established in vitro models. Our findings reveal that PHF6 levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of NOTCH1 and its downstream target genes. This functional interaction in developing thymocytes was confirmed in vivo using a phf6-deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced phf6 or notch1 activation. In summary, our work reveals that appropriate control of PHF6 expression is important for normal human hematopoiesis and provides clues towards the role of PHF6 in T-ALL development

A photoinduced pH jump applied to drug release from cucurbit[7]uril

A proof-of-principle for the application of a photoinduced pH jump for delivery of the Hoechst 33258 drug by disassembly of its host-guest complex with cucurbit[7]uril is described