CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Mechanisms for improving diabetes patient-provider communication through optimal use of e-clinical technologies.

Purpose: Effective health care and patient adherence to their prescribed regimens relies on successful communication between patients and their providers. This study examined mechanisms for optimizing patient-physician communication in subjects with type 2 diabetes, with a focus on optimizing the incorporation of e-clinical technology to improve engagement and communication. Methods: A total of 105 subjects with type 2 diabetes participating in a large US mode equivalency study were surveyed independently of this trial. In addition to demographic information, each subject was queried on their familiarity with and preference for e-clinical technologies. Survey questions focused on mobile technology use, perceptions, and preferences for improving communication and interactions with health care providers. Results: Subjects were diverse in age, sex, education, and ethnicity. Forty nine percent owned a smartphone, and 64% had a computer at home. Most subjects (81%) were interested in using electronic methods (eg, app on a smartphone, email, or text messages) to interact more with physicians between visits. The majority of subjects were interested in using technology to help manage their type 2 diabetes, including 62% favoring communicating with their health-care providers via email and a considerable fraction interested in using smartphones to be provided medication reminders (56%), clinical visit scheduling (55%), and text messaging (49%). Conclusion: Subjects are interested in using electronic methods to increase communication with their physicians and manage their type 2 diabetes. Health-care providers should consider engaging patients with e-clinical technology to increase patient-physician communication and for the ultimate goal of improved health care.

Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development.

Haematopoietic stem cells (HSCs) arise in the developing aorta during embryogenesis. The number of HSC clones born has been estimated through transplantation, but experimental approaches to assess the absolute number of forming HSCs in a native setting have remained challenging. Here, we applied single-cell and clonal analysis of HSCs in zebrafish to quantify developing HSCs. Targeting creERT2 in developing cd41:eGFP+ HSCs enabled long-term assessment of their blood contribution. We also applied the Brainbow-based multicolour Zebrabow system with drl:creERT2 that is active in early haematopoiesis to induce heritable colour barcoding unique to each HSC and its progeny. Our findings reveal that approximately 21 HSC clones exist prior to HSC emergence and 30 clones are present during peak production from aortic endothelium. Our methods further reveal that stress haematopoiesis, including sublethal irradiation and transplantation, reduces clonal diversity. Our findings provide quantitative insights into the early clonal events that regulate haematopoietic development.

Subjects with osteoarthritis can easily use a handheld touch screen electronic device to report medication use: qualitative results from a usability study.

OBJECTIVES: Electronic data capture is increasingly used to improve collection of patient-reported outcome measures in clinical trials and care. The validation of electronic patient-reported outcome devices requires information on patient preference and ease of use. This study conducted usability testing for a General Symptom Questionnaire and Medication Module™ on a handheld device for subjects with osteoarthritis (OA) to determine whether subjects can report on their symptoms and medication use using an electronic diary. METHODS: Nine subjects with OA participating in a large US mode equivalency study were surveyed independently in this study. Subjects completed a General Symptom Questionnaire and Medication Module™ using the LogPad® LW handheld device. Demographic and technology use information was collected, and the subjects were queried on device usability. RESULTS: Subjects reported that the handheld device was easy to use and that they were able to complete a General Symptom Questionnaire and Medication Module™ with little or no assistance. They did not report any issues with the screen or size of the device. Subjects were willing to travel with the device to complete electronic diaries at home or in public. Participants indicated that they would be able to use the handheld device to answer questions during a clinical trial. Subjects with OA experienced no physical discomfort during completion of either questionnaire. CONCLUSION: The General Symptom Questionnaire and Medication Module™ were usable and acceptable to subjects with OA on a handheld device. This was consistent regardless of previous experience and confidence with technology, despite the potential physical restrictions for an OA cohort.

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.

CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.

Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization. PAPERFLICK:

Getting more for your marrow: boosting hematopoietic stem cell numbers with PGE2.

Throughout the lifetime of an individual, hematopoietic stem cells (HSCs) self-renew and differentiate into lineages that include erythrocytes, platelets and all immune cells. HSC transplantation offers a potentially curative treatment for a number of hematopoietic and non-hematopoietic malignancies as well as immune and genetic disorders. Limited availability of immune-matched donors reduces the viable options for many patients in need of HSC transplantation, particularly those of diverse racial and ethnic backgrounds. Due to rapid availability and less stringent immune-matching requirements, umbilical cord blood (UCB) has emerged as a valuable source of transplantable HSCs. A single UCB unit contains a suboptimal number of HSCs for treating larger children or adults and there has thus been great clinical interest in expanding UCB HSCs ex vivo for use in transplantation. In this review we discuss the latest research and future avenues for the therapeutic use of small lipid mediator dmPGE2 to expand HSC numbers for transplantation. Originally identified in a chemical screen in zebrafish, dmPGE2 has now advanced to a phase II clinical trial as a therapy for patients with leukemia and lymphoma who are undergoing UCB transplantation.

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish.

The zebrafish has become a standard model system for stem cell and tissue regeneration research, based on powerful genetics, high tissue regenerative capacity and low maintenance costs. Yet, these studies can be challenged by current limitations of tissue visualization techniques in adult animals. Here we describe new imaging methodology and present several ubiquitous and tissue-specific luciferase-based transgenic lines, which we have termed zebraflash, that facilitate the assessment of regeneration and engraftment in freely moving adult zebrafish. We show that luciferase-based live imaging reliably estimates muscle quantity in an internal organ, the heart, and can longitudinally follow cardiac regeneration in individual animals after major injury. Furthermore, luciferase-based detection enables visualization and quantification of engraftment in live recipients of transplanted hematopoietic stem cell progeny, with advantages in sensitivity and gross spatial resolution over fluorescence detection. Our findings present a versatile resource for monitoring and dissecting vertebrate stem cell and regeneration biology.

In situ hybridization assay-based small molecule screening in zebrafish.

In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts.

Endostatin lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition.

Antiangiogenesis therapy has become a vital part of the armamentarium against cancer. Hypertension is a dose-limiting toxicity for VEGF inhibitors. Thus, there is a pressing need to address the associated adverse events so these agents can be better used. The hypertension may be mediated by reduced NO bioavailability resulting from VEGF inhibition. We proposed that the hypertension may be prevented by coadministration with endostatin (ES), an endogenous angiogenesis inhibitor with antitumor effects shown to increase endothelial NO production in vitro. We determined that Fc-conjugated ES promoted NO production in endothelial and smooth muscle cells. ES also lowered blood pressure in normotensive mice and prevented hypertension induced by anti-VEGF antibodies. This effect was associated with higher circulating nitrate levels and was absent in eNOS-knockout mice, implicating a NO-mediated mechanism. Retrospective study of patients treated with ES in a clinical trial revealed a small but significant reduction in blood pressure, suggesting that the findings may translate to the clinic. Coadministration of ES with VEGF inhibitors may offer a unique strategy to prevent drug-related hypertension and enhance antiangiogenic tumor suppression.

Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration.

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

Newly emerging roles for prostaglandin E2 regulation of hematopoiesis and hematopoietic stem cell engraftment.

PURPOSE OF REVIEW: Hematopoietic stem cell (HSC) transplantation is an effective treatment for leukemia, lymphoma, blood disorders, and autoimmune diseases. Successful transplantation is dependent upon efficient homing and engraftment of HSCs. Recently, prostaglandin E2 (PGE2) exposure, either in vivo or ex vivo, has been shown to increase engraftment. These results establish PGE2 as a regulator of hematopoietic development. RECENT FINDINGS: The underlying mechanisms of PGE2 regulation of HSC development were poorly understood until recently. Ex-vivo exposure of LSK cells to PGE2 results in increased homing efficiency of HSCs to the murine bone marrow compartment. In addition, in-vivo treatment with PGE2 preferentially expands short-term HSCs without affecting long-term HSC number and engraftment in murine bone marrow. PGE2 acts through EP4 receptors to mediate lymphoid precursor development in the zebrafish. An in-vivo interaction between PGE2 and the Wnt signaling pathways controls HSC engraftment. SUMMARY: PGE2 has a new role in HSC homing and survival, as well as short-term-HSC engraftment. PGE2 is currently being tested in clinical trials as a potential therapy to enhance HSC engraftment following a transplantation procedure.

Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy.

Angiopoietins were thought to be endothelial cell-specific via the tie2 receptor. We showed that angiopoietin-1 (ang1) also interacts with integrins on cardiac myocytes (CMs) to increase survival. Because ang1 monomers bind and activate integrins (not tie2), we determined their function in vivo. We examined monomer and multimer expressions during physiological and pathological cardiac remodeling and overexpressed ang1 monomers in phenylephrine-induced cardiac hypertrophy. Cardiac ang1 levels (mRNA, protein) increased during postnatal development and decreased with phenylephrine-induced cardiac hypertrophy, whereas tie2 phosphorylations were unchanged. We found that most or all of the changes during cardiac remodeling were in monomers, offering an explanation for unchanged tie2 activity. Heart tissue contains abundant ang1 monomers and few multimers (Western blotting). We generated plasmids that produce ang1 monomers (ang1-256), injected them into mice, and confirmed cardiac expression (immunohistochemistry, RT-PCR). Ang1 monomers localize to CMs, smooth muscle cells, and endothelial cells. In phenylephrine-induced cardiac hypertrophy, ang1-256 reduced left ventricle (LV)/tibia ratios, fetal gene expressions (atrial and brain natriuretic peptides, skeletal actin, beta-myosin heavy chain), and fibrosis (collagen III), and increased LV prosurvival signaling (akt, MAPK(p42/44)), and AMPK(T172). However, tie2 phosphorylations were unchanged. Ang1-256 increased integrin-linked kinase, a key regulator of integrin signaling and cardiac health. Collectively, these results suggest a role for ang1 monomers in cardiac remodeling.

Interpreter accuracy and informed consent among Spanish-speaking families with cancer.

Language interpreters mediate a growing number of health care communication events, including the informed consent process, which underlies the ethical conduct of clinical research. This article explores a key interpretive outcome, accuracy, in 21 Spanish/English informed consent consultations. Discussions were transcribed, translated, and coded according to established techniques. Most (74%) discussion was accurately interpreted (range: 47%-98%; std. deviation: .137). Accuracy was lower in the more technical portions of the discussion when compared with nontechnical portions such as discussion of coping and quality-of-life issues (p = .024). The concept of "randomization" often was poorly communicated and interpreted. These differences may be due in part to the use of long, uninterrupted, and jargon-filled sentences by clinicians explaining research, among other factors. The article concludes that accuracy may be promoted if clinicians used less technical language and shorter sentences, and are more "process driven." Interpreters may need to be better informed about research-related concepts such as randomization. Further research is needed to determine the effects of interpretive accuracy on the informed consent process, the decision to participate in research, and other outcomes.