Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification.

Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood. Transcription activator-like effectors (TALEs) are a novel class of genetic tool based on the modular DNA-binding domains of Xanthomonas TAL proteins, which enable DNA sequence-specific targeting and the manipulation of endogenous gene expression. Here, we report TALEs engineered to target the PU.1-14kb and Scl+40kb transcriptional enhancers as efficient new tools to perturb the expression of these key haematopoietic TFs. We confirmed the efficiency of these TALEs at the single-cell level using high-throughput RT-qPCR, which also allowed us to assess the consequences of both PU.1 activation and repression on wider TF networks during developmental haematopoiesis. Combined with comprehensive cellular assays, these experiments uncovered novel roles for PU.1 during early haematopoietic specification. Finally, transgenic mouse studies confirmed that the PU.1-14kb element is active at sites of definitive haematopoiesis in vivo and PU.1 is detectable in haemogenic endothelium and early committing blood cells. We therefore establish TALEs as powerful new tools to study the functionality of transcriptional networks that control developmental processes such as early haematopoiesis.

Risk mapping for better governance in biobanking: the case of biobank.cy.

Introduction: Risk governance is central for the successful and ethical operation of biobanks and the continued social license for being custodians of samples and data. Risks in biobanking are often framed as risks for participants, whereas the biobank's risks are often considered as technical ones. Risk governance relies on identifying, assessing, mitigating and communicating all risks based on technical and standardized procedures. However, within such processes, biobank staff are often involved tangentially. In this study, the aim has been to conduct a risk mapping exercise bringing biobank staff as key actors into the process, making better sense of emerging structure of biobanks. Methods: Based on the qualitative research method of situational analysis as well as the card-based discussion and stakeholder engagement processes, risk mapping was conducted at the biobank setting as an interactive engagement exercise. The analyzed material comprises mainly of moderated group discussions. Results: The findings from the risk mapping activity are framed through an organismic metaphor: the biobank as a growing, living organism in a changing environment, where trust and sustainability are cross-cutting elements in making sense of the risks. Focusing on the situatedness of the dynamics within biobanking activity highlights the importance of prioritizing relations at the core of risk governance and promoting ethicality in the biobanking process by expanding the repertoire of considered risks. Conclusion: With the organismic metaphor, the research brings the diverse group of biobank staff to the central stage for risk governance, highlighting how accounting for such diversity and interdependencies at the biobank setting is a prerequisite for an adaptive risk governance.

An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability.

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.

Redox chemistry of sulphate and uranium in a phosphogypsum tailings dump.

The present study aims to assess the effect of redox conditions existing within the tailings dump on the stability of phosphogypsum (e.g. sulphate reduction) and uranium(VI). Phosphogypsum sampling and in-situ measurements were carried out at a coastal tailings dump in Vasiliko Cyprus, pH, E(H) and solubility experiments were performed in simulated laboratory systems and thermodynamic calculations using MINTEQA2. Generally, in the open tailings dump oxidizing conditions predominate stabilizing sulphur and uranium in their hexavalent oxidation states. On the other hand, after the application of a soil/vegetative cover and in the presence of natural organic matter, anoxic conditions prevail (E(H) < -70 mV) resulting in S(VI) and U(VI) reduction to S(-II) and U(IV), respectively. Although, the sulphide anion can form very insoluble compounds with heavy metal ions (e.g. Cd(II), Pb(II) etc.) and U(IV) oxide has very low solubility, partial reduction of sulphate to sulphide within gypsum may affect the stability of phosphogypsum resulting in enhanced erosion of the material by rain- and seawater and washing out of contaminants in particulate/colloidal form.

Experimental and theoretical studies on physico-chemical parameters affecting the solubility of phosphogypsum.

Phosphogypsum is a waste by-product of the phosphate fertilizer industry, which is usually disposed in the environment because of its restricted use in industrial applications. Physico-chemical conditions existing in stack fluids and leachates are of major importance and determine solubility and redox stability of phosphogypsum, as well as radionuclide release from stacks to terrestrial environments. The aim of this study is to assess the effect of key parameters (e.g. ionic strength, temperature, pH) on the solubility of phosphogypsum. Phosphogypsum sampling and in-situ measurements were carried out at a coastal stack in Cyprus, solubility experiments were performed in simulated laboratory systems and thermodynamic calculations by means of MINTEQA2, an equilibrium speciation model. Generally, increasing ionic strength and temperature leads to increased phosphogypsum solubility, with the former being much more effective. The increased solubility of phosphogypsum in saline solutions is attributed solely to ionic strength effects on the activity of ionic species in solution and no solid phase transformations could be observed. The effect of pH on phosphogypsum solubility seems to be insignificant at least in a pH range between 4 and 8. Regarding uranium levels, there is a strong correlation between salinity and uranium concentration and linear correlation between phosphogypsum solubility and uranium levels in stack solutions, indicating the incorporation of uranium into the gypsum lattice and the formation of a solid solution.