XTcf-3 Transcription Factor Mediates β-Catenin-Induced Axis Formation in Xenopus Embryos

AbstractXTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to β-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of β-catenin. The β-catenin–XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (ΔN) abrogates the interaction with β-catenin, as well as the consequent transcription activation. This dominant-negative ΔN mutant suppresses the induction of axis duplication by microinjected β-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by β-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes

Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles

Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be non-random and represent relevant biology that may otherwise be missed, but also because they represent a significant fraction of the immunoprecipitated material, we designed a double-fragmentation ChIP-seq procedure. After conventional crosslinking and immunoprecipitation, chromatin is de-crosslinked and sheared a second time to concentrate fragments in the optimal size range for NGS. Besides the benefits of increased chromatin yields, the procedure also eliminates a laborious size-selection step. We show that the double-fragmentation ChIP-seq approach allows for the generation of biologically relevant genome-wide protein-DNA binding profiles from sub-nanogram amounts of TCF7L2/TCF4, TBP and H3K4me3 immunoprecipitated material. Although optimized for the AB/SOLiD platform, the same approach may be applied to other platforms

Streptomyces‐derived quorum‐sensing systems engineered for adjustable transgene expression in mammalian cells and mice

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene‐function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum‐sensing to coordinate population‐wide responses to physiological and/or physicochemical signals. A generic bacterial quorum‐sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon. In Streptomyces, a family of butyrolactones and their corresponding receptor proteins, serve as quorum‐sensing systems that control morphological development and antibiotic biosynthesis. Fusion of the Streptomyces coelicolor quorum‐sensing receptor (ScbR) to a eukaryotic transactivation domain (VP16) created a mammalian transactivator (SCA) which binds and adjusts transcription from chimeric promoters containing an SCA‐specific operator module (PSPA). Expression of erythropoietin or the human secreted alkaline phosphatase (SEAP) by this quorum‐sensor‐regulated gene expression system (QuoRex) could be fine‐tuned by non‐toxic butyrolactones in a variety of mammalian cells including human primary and mouse embryonic stem cells. Following intraperitoneal implantation of microencapsulated Chinese hamster ovary cells transgenic for QuoRex‐controlled SEAP expression into mice, the serum levels of this model glycoprotein could be adjusted to desired concentrations using different butyrolactone dosing regime

The role of tunneling in enzyme catalysis of C–H activation

AbstractRecent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C–H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects

Long-term culture of genome-stable bipotent stem cells from adult human liver.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.This work was supported by grants to MH (EU/236954) and to HC (The United European Gastroenterology Federation (UEGF) Research Prize 2010, EU/232814-StemCellMark and NWO/116002008). MH is supported by The Wellcome Trust Sir Henry Dale fellowship. The Rspo cell line was kindly provided by Dr. Calvin Kuo.This is the final published version. It first appeared at http://www.cell.com/abstract/S0092-8674%2814%2901566-9

Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.This work was supported by NIRM/ Clevers and Stichting Vrienden van het Hubrecht (O.B.), EU/232814-StemCellMark and Skolkovo 077 MPA (J.H.v.E.), NIH/MIT Subaward 5710002735 (to D.E.S.), KWF/PF-HUBR 2007-3956 and Stichting Vrienden van het Hubrecht (M.v.d.W.), European Research Council Advanced Grant ERC-AdG 294325-GeneNoiseControl (to K.W. and A.v.O.), and Wellcome Trust Grant 098357/Z/12/Z (to B.D.S.)

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions

A paired-kidney allocation study found superior survival with HLA-DR compatible kidney transplants in the Eurotransplant Senior Program

The Eurotransplant Senior Program (ESP) has expedited the chance for elderly patients with kidney failure to receive a timely transplant. This current study evaluated survival parameters of kidneys donated after brain death with or without matching for HLA-DR antigens. This cohort study evaluated the period within ESP with paired allocation of 675 kidneys from donors 65 years and older to transplant candidates 65 years and older, the first kidney to 341 patients within the Eurotransplant Senior DR-compatible Program and 334 contralateral kidneys without (ESP) HLA-DR antigen matching. We used Kaplan-Meier estimates and competing risk analysis to assess all cause mortality and kidney graft failure, respectively. The log-rank test and Cox proportional hazards regression were used for comparisons. Within ESP, matching for HLA-DR antigens was associated with a significantly lower five-year risk of mortality (hazard ratio 0.71; 95% confidence interval 0.53-0.95) and significantly lower cause-specific hazards for kidney graft failure and return to dialysis at one year (0.55; 0.35-0.87) and five years (0.73; 0.53-0.99) post-transplant. Allocation based on HLA-DR matching resulted in longer cold ischemia (mean difference 1.00 hours; 95% confidence interval: 0.32-1.68) and kidney offers with a significantly shorter median dialysis vintage of 2.4 versus 4.1 yrs. in ESP without matching. Thus, our allocation based on HLA-DR matching improved five-year patient and kidney allograft survival. Hence, our paired allocation study suggests a superior outcome of HLA-DR matching in the context of old-for-old kidney transplantation.</p

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions