N-terminal and core-domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance

AbstractRandom mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds

Tying Up a Loose End: On the Role of the C‐Terminal CCHHRAG Fragment of the Metalloregulator CueR

The transcriptional regulator CueR is activated by the binding of CuI, AgI, or AuI to two cysteinates in a near-linear fashion. The C-terminal CCHHRAG sequence in Escherichia coli CueR present potential additional metal binding ligands and here we explore the effect of deleting this fragment on the binding of AgI to CueR. CD spectroscopic and ESI-MS data indicate that the high AgI-binding affinity of WT-CueR is significantly reduced in Δ7C-CueR.[111 Ag PAC spectroscopy demonstrates that the WT-CueR metal site structure (AgS2) is conserved, but less populated in the truncated variant. Thus, the function of the C-terminal fragment may be to stabilize the two-coordinate metal site for cognate monovalent metal ions. In a broader perspective this is an example of residues beyond the second coordination sphere affecting metal site physicochemical properties while leaving the structure unperturbed

Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation

YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration.

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.This work was supported by Worldwide Cancer Research (13-1216 to KBJ), Lundbeck Foundation (R105-A9755 to KBJ), the Danish Cancer Society (R56-A2907 and R124-A7724 to KBJ), the Carlsberg Foundation (to KBJ), EMBO Young Investigator programme (to KBJ), AIRC Special Program Molecular Clinical Oncology ‘‘5 per mille’’ (to SP), an AIRC PI-Grant (to SP), Epigenetics Flagship projects (CNR-Miur grants. to SP), the DFF mobilix programme (to SY), Marie Curie fellowship programme (SY and JG), Foundation of Aase and Ejnar Danielsen (OHN), Axel Muusfeldts Foundation (OHN), The Ragnar Söderberg Foundation (CDM). This project has received funding from the European Union’s Horizon 2020 research and innovation programme (grant agreements STEMHEALTH ERCCoG682665 and INTENS 668294 to KBJ and DENOVOSTEM No. 670126 to SP)

YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element

Canine specific ELISA for coagulation factor VII

Canine coagulation factor VII (FVII) deficiency can be hereditary or acquired and may cause life threatening bleeding episodes if untreated. FVII procoagulant activity can be measured by FVII activity (FVII:C), but assays for measurement of canine specific FVII antigen (FVII:Ag) have not been available to date. In this study, a canine specific ELISA for measurement of FVII:Ag in plasma was developed and validated. The FVII:Ag ELISA correctly diagnosed homozygous and heterozygous hereditary FVII deficiency. Together with activity based assays, such as FVII:C, the FVII:Ag ELISA should be valuable in the diagnosis of hereditary canine FVII deficiency

Case management used to optimize cancer care pathways: A systematic review

<p>Abstract</p> <p>Background</p> <p>Reports of inadequate cancer patient care have given rise to various interventions to support cancer care pathways which, overall, seem poorly studied. Case management (CM) is one method that may support a cost-effective, high-quality patient-centred treatment and care.</p> <p>The purpose of this article was to summarise intervention characteristics, outcomes of interest, results, and validity components of the published randomized controlled trials (RCTs) examining CM as a method for optimizing cancer care pathways.</p> <p>Methods</p> <p>PubMed, Embase, Web of Science, CINAHL and The Cochrane Central Register of Controlled Trials were systematically searched for RCTs published all years up to August 2008. Identified papers were included if they passed the following standards. Inclusion criteria: 1) The intervention should meet the criteria for CM which includes multidisciplinary collaboration, care co-ordination, and it should include in-person meetings between patient and the case manager aimed at supporting, informing and educating the patient. 2) The intervention should focus on cancer patient care. 3) The intervention should aim to improve subjective or objective quality outcomes, and effects should be reported in the paper.</p> <p>Exclusion criteria: Studies centred on cancer screening or palliative cancer care.</p> <p>Data extraction was conducted in order to obtain a descriptive overview of intervention characteristics, outcomes of interest and findings. Elements of CONSORT guidelines and checklists were used to assess aspects of study validity.</p> <p>Results</p> <p>The searches identified 654 unique papers, of which 25 were retrieved for scrutiny. Seven papers were finally included. Intervention characteristics, outcomes studied, findings and methodological aspects were all very diverse.</p> <p>Conclusion</p> <p>Due to the scarcity of papers included (seven), significant heterogeneity in target group, intervention setting, outcomes measured and methodologies applied, no conclusions can be drawn about the effect of CM on cancer patient care.</p> <p>It is a major challenge that CM shrouds in a "black box", which means that it is difficult to determine which aspect(s) of interventions contribute to overall effects. More trials on rigorously developed CM interventions (opening up the "black box") are needed as is the re-testing of interventions and outcomes studied in various settings.</p

The Coevolution of Virulence: Tolerance in Perspective

Coevolutionary interactions, such as those between host and parasite, predator and prey, or plant and pollinator, evolve subject to the genes of both interactors. It is clear, for example, that the evolution of pollination strategies can only be understood with knowledge of both the pollinator and the pollinated. Studies of the evolution of virulence, the reduction in host fitness due to infection, have nonetheless tended to focus on parasite evolution. Host-centric approaches have also been proposed—for example, under the rubric of “tolerance”, the ability of hosts to minimize virulence without necessarily minimizing parasite density. Within the tolerance framework, however, there is room for more comprehensive measures of host fitness traits, and for fuller consideration of the consequences of coevolution. For example, the evolution of tolerance can result in changed selection on parasite populations, which should provoke parasite evolution despite the fact that tolerance is not directly antagonistic to parasite fitness. As a result, consideration of the potential for parasite counter-adaptation to host tolerance—whether evolved or medially manipulated—is essential to the emergence of a cohesive theory of biotic partnerships and robust disease control strategies