text:
(confirmed after 28 July)
Sunday, 28 August 2016
Time change: Revised to 10:45 - 12:45
Lunch on own
Sunday, 28 August 2016
Time change: 12:45 - 14:00
Time change: Revised to 14:00 - 15:45
1018 – Session Speaker Cancelled
Richard Van Etten, UC Irvine
Central role for ikaros in pre-b cell differentiation and pathogenesis of high-risk b-cell acute lymphoblastic leukemia
1033 – Session Speaker Added
Danica Chen, University of California, Berkeley, USA
Mitochondrial UPR-mediated metabolic checkpoint, hematopoietic stem cell aging and rejuvenation
Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in hematopoietic stem cell (HSC) biology highlight a mitochondrial UPR-mediated metabolic checkpoint that is essential for HSCs to return to the quiescent state. As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced and mitochondrial oxidative stress is increased, which is associated with increased mitochondrial protein folding stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions.
New Investigators Technology Session and Lunch*
Bertie Gottgens, University of Cambridge Suffolk, UK
Doug Hinerfeld, Fluidigm, USA
New Investigators Career Session and Lunch*
Mervin C. Yoder, Indiana University - Wells Center for Pediatric Research Indianapolis, IN
Sheila Chari, Cell Stem Cell, Cambridge, MA, USA
Keith Humphries, Experimental Hematology, Vancouver, BC, Canada
Terry Bishop, NIDDK/NIH Bethesda, MD, USA
*presenters added
3001 has declined the presentation invitation.
3002 has declined the presentation invitation.
3004 has declined the presentation invitation.
3014 has declined the presentation invitation.
3038 has declined the presentation invitation.
3047 has declined the presentation invitation.
3053 has declined the presentation invitation.
3075 has declined the presentation invitation.
3079 has declined the presentation invitation.
3097 has declined the presentation invitation.
3108 has declined the presentation invitation.
3111 has declined the presentation invitation.
3112 has declined the presentation invitation.
3114 additional authors are Allison Superneau and Glen Lowery.
3116 has declined the presentation invitation.
3123 has declined the presentation invitation.
3130 has declined the presentation invitation.
3132 has declined the presentation invitation.
3139 has declined the presentation invitation.
3140 has declined the presentation invitation.
3159 has declined the presentation invitation.
3170 has declined the presentation invitation.
3179 presenter has changed from Samanta A. Mariani to Chris Vink
3181 has declined the presentation invitation.
3193 has declined the presentation invitation.
Updated Abstracts
3093 author order has changed to Gudmundur Norddahl, Tobias Maetzig, Patricia Rosten
Saturday, 27 August 2016
13:00 - 14:45
2015 – The spliceosomal SRSF2P95H mutation promotes myeloid biased
Carl Walkley, St. Vincent Institute, Australia
Carl Walkley, Monique Smeets, Shuh Ying Tan, Jane Xu, Alistair Chalk, Meaghan Wall, Louise Purton
St Vincent's Institute
Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are hematological malignancies with limited treatment options. In MDS and CMML there are high rates of mutations in components of the RNA splicing machinery. Of the spliceosomal genes, 12-15% of MDS and 28-52% of CMML patients carry mutations in SRSF2, predominantly involving proline 95 (P95). Mutation of SRSF2 is an independent poor risk marker for overall survival and for progression to AML. It is largely uncharacterised how SRSF2P95 mutations impact on hematopoiesis and lead to both MDS and CMML. A recently reported conditional Srsf2P95H mutant mouse model did not develop features of advanced MDS (Kim et al, Cancer Cell 2015), nor did it display myeloid bias or positive selection for SRSF2 mutant cells, as has been reported in humans. We have generated a conditionally activatable Srsf2P95H model where the mutation is expressed from the endogenous locus in a heterozygous manner. Using ubiquitous (R26-CreER) or HSC specific (hScl-CreER) models to activate expression, we see macrocytic anemia and a mild leucocytosis resulting from elevated neutrophils. Analysis of bone marrow demonstrates an increase in frequencies of myeloid cells at the expense of erythroid- and B-cells. There is reduced expression of CD45RB, direct evidence of altered splicing in vivo. Bone marrow transplantation 20-24 weeks after Srsf2P95H expression resulted in a profound myeloid biased differentiation of the mutant cells. There is progressive loss of chimerism with cells isolated from the R26-CreER model, which is not seen with the HSC specific activation model, suggestive of differences in the impact of Srsf2P95H mutation depending on where it is expressed. At ~12 months post-tamoxifen, macrocytic anemia and myeloid dysplasia are seen in the PB of Srsf2P95H/+ animals, core characteristics of MDS. BM cellularity is largely preserved but with a profound myeloid bias (>70% BM granulocytes). In summary, these animals display a profound disruption in BM hematopoiesis. Abnormal phenotypes are seen in both the R26-CreER and hScl-CreER models. The changes are consistent with human MDS with SRSF2P95 mutation. Our results demonstrate that expression of SRSF2P95H from its endogenous locus is sufficient to initiate MDS and establishes a model that recapitulates fundamental features of human.
Saturday, 27 August 2016
13:00 - 14:45
2017 – Using chromatin profiling to identify signaling-responsive transcriptional regulators of hematopoietic specification
Constanze Bonifer, University of Birmingham, USA
Constanze Bonifer1, Nadine Obier1 , Pierre Cauchy1, Jane Gilmour1, Salam Assi1, Michael Lie-A-Ling2, Debbie Goode3, Berthold Goettgens3, Georges Lacaud2, Valerie Kouskoff2
1University of Birmingham, 2University of Manchester, 3University of Cambridge
It is unclear how specific transcriptional networks respond to outside signals to direct developmental stage-specific gene expression. The reason for this is the fact that signaling pathways consist of a myriad of components operating in a cell type-specific fashion and displaying multiple types of cross-talk making this process difficult to study. However, the response to signals is mediated by inducible transcription factors which bind to their cognate cis-regulatory elements, and thus the response to signaling is hard-wired into DNA. By identifying and studying the function of such sequences and the factors binding to them we can obtain first insights into how signal transduction processes are coordinated at the genomic level and then work our way backwards. We have previously identified transcriptional networks driving hematopoietic specification in differentiating embryonic stem cells (Goode et al., Dev Cell 2016). To identify signaling responsive factors interacting with this network, we used DNaseI hypersensitive site mapping and digital footprinting. This approach identified motifs for such factors enriched in cis-regulatory elements playing a role at specific stages of hematopoietic specification and highlighted motifs for TEAD factors as mediators of Hippo signaling, and AP-1 family members as mediators of MAP-Kinase signaling. By targeting these transcription factors directly combined with the identification of their global binding sites in cells representing different stages of hematopoietic specification, we show that (i) TEAD and YAP interaction is essential for hematopoietic development, (ii) AP-1 regulates the balance between the vascular and the hematopoietic program, (iii) AP-1 is required for the binding and expression of TEAD4 at a sub-set of genes in the hemogenic endothelium involved in vasculogenesis, (iv) that after the endothelial-hematopoietic transition cis-regulatory element activity is uncoupled from Hippo signaling and finally, that (v) AP-1 activity is required for myelopoiesis, adding to the activity of cis-elements binding a different set of transcription factors. Our work provides a global view of how signaling responses are integrated at the genomic level and how signaling responsive transcription factors interact with constitutive factors to program the chromatin landscape in response to the environment.
Sunday, 28 August 2016
09:00 - 10:30
Session 11B: MOLECULAR CONTROL III
2033 - Eicosanoid activation of GPCR signaling enhances hematopoiesis and marrow transplant
Jamie L. Lahvic, Harvard Medical School, USA
Jamie L. Lahvic1, Michelle Ammerman2, Pulin Li3, Song Yang2, Nan Chiang1, 4, Paul Norris1, 4, Michael Chase2, Olivia Weis2, Yi Zhou1,2, 5, Charles Serhan1, 4, Leonard Zon1, 2,4,5
1Harvard Stem Cell Institute, 2Boston Children's Hospital, 3California Institute of Technology, 4Brigham and Women's Hospital, 5Howard Hughes Medical Institute
Bioactive lipids play an important role as signaling molecules both during embryo development and adult tissue homeostasis. However, due to their small-molecule nature, identifying their receptors biochemically has been a long-standing challenge which impedes the understanding of the biological processes they regulate. Previously, we discovered that 11,12-epoxyeicosatrienoic acid (EET) could promote hematopoietic stem and progenitor cells (HSPC) engraftment in adult marrow transplant, as well as specification of HSPC from hemogenic endothelium. However, the identity of its receptor remains unknown despite more than a decade of research. Here, we utilized a novel bioinformatic approach to identify candidate EET receptors and identified a candidate functional in cull culture, zebrafish and mouse assays. We performed transcriptomic profiling on three human cell lines with clear EET-responsive phenotypes and found 37 GPCR expressed in common. 27 of these GPCR were also expressed in a non-EET-responsive cell line, leaving 10 candidate GPCR. Of these, only GPR132 exhibited EET-dependent recruitment of β-arrestin. GPR132 was previously identified as a receptor for a variety of small oxygenated fatty acids, which we confirmed by β-arrestin assay. Like EET, these GPR132 ligands increased HSPC numbers in the zebrafish aorta-gonad- mesonephros (AGM) and caused ectopic expression of the HSPC marker runx1 in the zebrafish tail, a phenotype that was previously seen only with EET treatment. GPR132 is required for EET phenotypes; morpholino knockdown of GPR132 prevented the EET-induced increase of runx1 in both the AGM and tail. Finally, we performed competitive whole bone marrow transplant using wildtype and GPR132-/- mice as donors and found that while treatment with EET increases engraftment of WT donor cells, no such improvement is seen in GPR132-/- cells. Combining bioinformatic, biochemical, and genetic approaches, we identified GPR132 as a receptor for EET and other small oxygenated fatty acids responsible for regulating hematopoiesis and marrow transplant. Genetic manipulation of GPR132 could help illuminate the endogenous roles of its fatty acid ligands.
Antoniana Batsivari1; Stanislav Rybtsov2; Celine Souilhol2; Anahi Binagui-Casas2; Lucia Morgado-Palacin2; David Hills2; Suling Zhao2; Alexander Medvinsky2
1MRC Centre for Regenerative Medicine, University of Edinburgh, United Kingdom; 2MRC Centre for Regenerative Medicine, University of Edinburgh, UK
Definitive haematopoietic stem cells (dHSC), capable of giving rise to all blood cell types, are generated intra-embryonically in the aorta-gonad-mesonephros (AGM) region of the mid-gestation embryo. Recent reports revealed that HSCs emerge through multistep maturation of precursors: proHSC--> preHSC I--> preHSC II--> dHSC. These haematopoietic precursors are found in the intra-aortic clusters, which bud from the aortic endothelium. Proliferation is a critical process for haematopoiesis as its regulation influences the balance between HSC self-renewal and differentiation. While proliferation of foetal and adult HSCs has been intensely studied, the cell cycle status of embryonic HSCs and their precursors is poorly characterised.
Here we used Fucci reporter mice that enable in vivo visualisation of cell cycle in combination with in vivo functional assays to assess the cell cycle status of preHSCs at different developmental stages. Our analysis shows that at E9.5 proHSCs are slowly cycling (G0/G1), while at E10.5 the emerging preHSC I are actively cycling, suggesting an expansion phase at this stage. Interestingly, one day later, the cell cycle of preHSC I slow down, while the emerging preHSC II are actively cycling. Finally, at E11.5, the dHSCs are found in G0/G1phases, like their foetal liver counterparts. Furthermore, we demonstrate by phenotypic analysis that the slowly cycling precursors show low levels of c-Kit expression and are predominantly closely attached to the endothelium, whereas the mature actively cycling cells express c-Kit highly and are at the top of the cluster.
Collectively these data suggest that transition from one HSC precursor type to another is concomitant with changes in cell cycle profile as well as localisation. Moreover, our data reveal important heterogeneity in cell cycle of preHSCs and suggest a role of c-Kit/SCF signalling pathway in expansion of haematopoietic cells.
Amy Bradburn1; Kevin Rouault-Pierre2; Dominique Bonnet2
1Francis Crick Institute, London, United Kingdom; 2Francis Crick Institute
Chemoresistance and relapse are currently the biggest problems facing treatment of AML, with retention of leukaemia initiating cells (LIC) post therapy believed to be a main contributor. Standard AML chemotherapy regimes utilize cytarabine, a cytotoxic compound inflicting DNA damage in proliferating cells and inducing cell death. Cancer cells often have a deficiency in DNA repair pathways, which sensitize them to such treatments. Comparing normal and malignant haematopoietic cells in their response to cytarabine will determine whether differing responses to DNA damage and activation of DNA repair mechanisms contribute to chemoresistance. Moreover, DNA repair in primitive human haematopoiesis and LIC's is still not well established.
Using AML cell lines, cord blood and CD34+ AML patient samples, a characterization of the DNA damage response post cytarabine treatment was performed. In vitro, cell lines were shown to have a range of sensitivities. Analysis of γH2AX foci formationshowed a significant increase in DNA damage after cytarabine exposure, however cell lines resistant to cytarabine, were quicker to recover their DNA damage, as shown by comet assay. As well, there was a faster revival of cell numbers and reduction in apoptosis. Similar findings were also observed when using ionizing radiation as a source of DNA damage. Analysis of the DNA repair mechanisms non-homologous end joining (NHEJ) and homologous recombination (HR) by 53BP1 and RAD51 localization respectively, showed NHEJ was preferentially used in response to cytarabine, with low rates of HR.
Normal cord blood haematopoietic stem (CD34+/CD38-) and progenitor (CD34+/CD38+) populations were compared with leukaemia initiating and tumour populations of CD34+ AML patient samples in vivo. All samples gained an increase in γH2AX foci and apoptosis post cytarabine exposure. Progenitor and tumour populations were most profoundly hit with a sparing of the more quiescent stem compartments. AML samples gave a heterogeneous response overall. NHEJ was primarily observed, however some samples showed increased localization of RAD51 compared to normal, which suggests enhanced HR contributes to response of AML to cytarabine.
Overall we see evidence of DNA repair in response to cytarabine treatment with leukaemic cells activating their repair mechanisms at a greater rate than normal haematopoietic cells.
Raedun Clarke1; Matthieu Bauer2; Jeffrey Sasaki2; Brian Groff2; Ryan Bjordahl2; Ramzey Abujarour2; Greg Bonello2; Tom Lee2; Weijie Lan2; Heather Foster2; Chris Lynn2; Dave Robbins2; Betsy Rezner2; Dan Shoemaker2; Bob Valamehr2
1CA, San Diego, United States; 2Fate Therapeutics Inc
Human induced pluripotent stem cell (hiPSC) technology has advanced into clinical trials, garnishing hope and excitement as an unlimited source of universally applicable cellular therapeutics. To advance hiPSCs as "off-the-shelf" hematopoietic cell-based immunotherapies it is essential to demonstrate a hiPSC platform with enhanced potency through genome-engineering of persistence, targeting and safety mechanisms while retaining the capacity to efficiently and reproducibly generate the diverse repertoire of immune effectors in a robust and scalable process. We have previously demonstrated that our reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables efficient multi-gene and multi-loci targeted engineering at a single cell level, in both nuclease-dependent and -independent strategies. Through genetic modifications at the single cell hiPSC stage, we demonstrate integration of inducible caspase-mediated and herpes simplex virus thymidine kinase suicide genes each into a unique safe harbor locus and increased effector cell persistence and immune evasion through HLA-related genetic modifications. Additionally we have developed a novel differentiation platform to derive hematopoietic cells from genetically enhanced hiPSCs in a highly scalable manner, void of an embryoid body intermediate, under serum- and feeder-free conditions. This platform represents a well-defined, small molecule-driven, staged protocol that can readily be translated into cGMP (current good manufacturing practice) settings. The derived hematopoietic population is definitive in nature as determine by Notch dependency and exhibits multi-lineage potential, as demonstrated through the formation of functional effector lymphocytes. These derived lymphocytes, including T and NK cells, are under investigation for in vitro and in vivo effector function including target specific recognition and enhanced killing potential. Hematopoietic cells generated can be successfully cryopreserved and banked, serving as a highly-stable feedstock for subsequent derivation of various cell types for therapeutic use. Our study indicates that naive hiPSCs are an ideal source for "off-the-shelf" hematopoietic cell-based immunotherapies.
Ashlee Conway
Monash University
Rationale: Congenital haemolytic anaemias comprise of a collection of severe, often fatal, genetic disorders of the red cell. TPI deficiency is a rare red cell disease caused by a genetic mutation in the enzyme TPI-1, resulting in haemolytic anaemia, splenomegaly, paralysis, and ultimately death in early childhood. No treatments exist for TPI deficiency, but animal models such as the Drosophila have previously shown that enzyme replacement therapy is a viable long-term solution. Adapting this therapy to a mammalian model of this haemolytic disease would provide the foundations for new clinical approaches towards TPI deficiency and similar congenital anaemias.
Aim: Using a mutagenised murine model of TPI deficiency, called RBC19, we hope to demonstrate the first mammalian bone marrow-derived enzyme replacement therapy as a plausible clinically applicable treatment for human TPI deficiency.
Methods: The RBC19 mutant mouse line was generated on an SJL/J background using ENU mutagenesis. G1 progeny were interbred to generate recessive G2 mutants, which were screened for changes in red cell volume (MCV). RBC19 mice were subject to whole exome sequencing (WES), which isolated a phenylalanine to serine base-pair mutation in amino acid 57 of the tpi-1 gene. Enzyme-replacement therapy was conducted using 1x106 bone marrow cells harvested from adult SJL/L wildtype femurs, which were intravenously transplanted into lethally irradiated RBC19 homozygous recipients.
Results: RBC19 homozygous mice were found to have an elevated MCV compared to SJL/J wildtypes (62.1fL to 43.3fL), as well as splenomegaly, reticulocytosis, and a reduced red cell half-life (44% of wildtype). The F57S mutation in the tpi-1 gene was shown to cause TPI protein instability and reduced enzyme activity (29% of wildtype). Following 6 weeks recovery from bone marrow transplantation, RBC19 recipients showed normalised MCV (45.3fL), reduced reticulocytes, and a normalised red cell half-life when compared to SJL controls.
Conclusions: ENU mutagenesis has generated a viable homozygous murine model that accurately recapitulates TPI deficiency with genetic, enzymatic, and haematological parallels to the human disease. This is the first instance in which enzyme replacement therapy has been adapted to a mammalian model of TPI deficiency, and has demonstrated that this red cell disease can be effectively treated long-term using bone marrow transplantation. The data obtained from these investigations provide the foundations for clinically translatable treatment of human TPI deficiency and similar congenital haemolytic anaemias.
Christina Eich1; Jochen Arlt2; Chris Vink3; Parham Solaimani Kartalaei4; Polynikis Kaimakis4; Wiggert A, van Cappellen5; Reinier van der Linden4; Elaine Dzierzak3
1Erasmus MC, Dep. of Cell Biology; 2University of Edinburgh, School of Physics and Astronomy; 3University of
Edinburgh, Centre for Inflammation Research, Queens Medical Research Institute; 4Erasmus Medical Center, Stem Cell Institute, Department of Cell Biology; 5Erasmus Medical Center, Department of Pathology
Underpinning cell behavior during development and differentiation are fundamental dynamic changes that drive cell fate processes away from a pre-existing equilibrium. The Gata2 transcription factor is an important regulator of blood cell differentiation and development, and is pivotal to hematopoietic stem cell generation (HSC). Within a short window of developmental time, HSCs originate from a natural differentiation of hemogenic endothelial cells through a process called endothelial to hematopoietic transition (EHT). Genetic studies in mice show that Gata2 is required for EHT and HSC generation, and that this process is very sensitive to reductions in Gata2 dosage. Surprisingly, nothing is known about the in vivo behavior of Gata2 expression in single cells during development, and particularly during EHT. In this study, we used a recently published Gata2-Venus reporter system with unperturbed Gata2 expression (Kaimakis et al, Blood, 2016) to follow Gata2 expression in single cells of the mouse midgestation aorta (the site of EHT and HSC generation) by vital confocal time-lapse imaging. EHT subsets that included single hemogenic endothelial cells, bulging cells and cluster cells were imaged over a 10 hours period. To quantify Gata2 levels, oscillation periodicity and amplitude, we developed a novel automated data processing methodology. EHT cell subsets could be distinguished by Gata2 expression levels and oscillation characteristics, with the greatest magnitude of Gata2 oscillation during EHT occurring in bulging cells. In Gata2 haploinsufficient embryos, which have greatly reduced HSC quantity and quality, Gata2 expression levels and oscillation characteristics in EHT cell subsets were profoundly disturbed. These single cell vital imaging results demonstrate a correlation between Gata2 oscillations, cell differentiation and acquisition of hematopoietic function during EHT, strongly suggesting that Gata2 oscillatory states and levels drive hematopoietic fate.
Elizabeth Ng1; Lisa Azzola2; Freya Bruveris2; Vincenzo Calvanese3; Belinda Phipson2;Katerina Vlahos2; Claire Hirst4; Vanta Jokubaitis5; Qing Yu6; Jovana Maksimovic2; Simone Liebscher7; Vania Januar2; Zhen Zhang8; Brenda Williams8; Aude Conscience4; Jennifer Durnall2; Steven Jackson9; Magdaline Costa4; David Elliott2; David Haylock8; Susan Nilsson8; Richard Saffery2; Katja Schenke-Layland7; Alicia Oshlack2; Hanna Mikkola10; Edouard Stanley2; Andrew Elefanty2
1Murdoch Childrens Research Institute ; 2Murdoch Childrens Research Institute; 3IUniversity of California, Los Angeles; 4Monash University; 5University of Melbourne; 6Shanghai Institutes for Biological Sciences; 7Eberhard Karls University Tübingen; 8Commonwealth Scientific and Industrial Research Organisation; 9University of Wisconsin-Madison; 10University of California, Los Angeles
The production of definitive hematopoietic stem/progenitor cells from human pluripotent stem cells (hPSCs) remains a significant challenge. Compared to repopulation-competent cord blood CD34+ cells, hPSC-derived CD34+ progenitors lackedHOXA gene expression, indicating incorrect mesoderm patterning. Using reporter lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition, we show that simultaneous modulation of WNT and ACTIVIN signaling yields hematopoietic progenitors with HOXA codes that more closely resemble those of cord blood. These cultures generate a network of aorta-like SOX17+ vessels, from which RUNX1C+ blood cells emerge - reminiscent of definitive hemogenesis within the aorta-gonad-mesonephros (AGM). Transcriptional profiles of nascent hematopoietic progenitors and corresponding cells sorted from human AGM displayed striking similarity in expression of cell surface receptors, signaling molecules and transcription factors. Our findings argue that HOXA codes are an indicator of mesoderm patterning, providing a rational and tractable readout for optimizing the generation of definitive hematopoietic lineages from hPSCs.
Eugenia Flores-Figueroa1; Alicia Aguilar-Navarro2; Dita Gratzinger3; A Berenice Meza-León2;Hector Mayani4; Ricardo Esquivel-Gómez5
1National Medical Center, IMSS., Mexico, Mexico; 2National Medical Center, IMSS; 3Stanford; 4National Medical Center, IMSS.; 5Orthopedics Hospital, IMSS.
Clonal hematopoiesis is a precursor to myeloid neoplasia, and aging is a factor that increases the risk of both. To date, we have not fully understood the mechanism that triggers clonal hematopoiesis within the bone marrow and the role of aging in this process, but we hypothesize that mesenchymal stromal cells (MSCs) may play an important role. MSCs contribute to development of myeloid neoplasia in mouse models and harbor chromosomal abnormalities in some hematopoietic neoplasms. The vast majority of CD34+ cells are in intimate contact with CD271 MSC in intact bone marrow core biopsies, and CD271+ MSC density is an independent prognosis factor in MDS, Little data exists regarding the functional and phenotypic properties of human MSCs with aging. The aim of the study was to compare the functional properties of MSCs and frequency of hematopoietic stem and progenitor cells in bone marrow samples from healthy adults (40-60 years) and healthy older adults (61-89 years) in the prime age range at risk for myelodysplasia and acute myeloid leukemia.
Methods: Bone marrow samples were obtained from otherwise healthy adults undergoing hip replacement surgery (n=23)(institutional ethical approval R-2012-785-092), from adults (40 to 60 years) and older adults (61 to 89 years). Flow cytometry was performed using the following markers: CD45, low affinity NGFR (CD271) and CD90, CD34, CD38, CD49f and lineage. MSC clonogenic potential was evaluated by the CFU-F assay. MSC potential to differentiate into adipocytes was evaluated by oil red O staining, and osteogenic differentiation by Von Kossa staining.
Results: The clonogenic capacity of MSC was significantly lower in the older adult group as compared to the adult group (3.1±1 vs 1.5±1.2 p=0.03). Among older adults differentiation potential was intact towards the adipogenic lineage, but increased towards osteoblasts (p=0.03 chi-square test). When we analyzed the adults and older adults groups pooled together, we found a strong correlation between the frequency of CD271+CD90+lin- MSC and CD34+CD38-lin- hematopoietic progenitor/stem cells (HPSC) (p=0.0055) but not to CD34+CD38-CD90+CD49f+lin- hematopoietic stem cells (HSC) or more committed CD34+CD38+ hematopoietic progenitor cells (HPC).
Conclusions: We have found decreases in the clonogenic capacity and changes in the differentiation potential of MSCs among older adults in the age range at increased risk of myeloid neoplasia. Among adults and older adults HPSC but not HSC or committed HPC frequency correlates with MSC frequency. The combination of functional changes in MSC with aging and the correlation of MSC frequency with HPSC frequency suggests that changes in the myeloid compartment among older adults might be related to MSC aging. Funding. Conacyt (CB- 2012-01/19947)
Xiang Li1; Sijie Zhang2; Ganglong Yang2; Zengqi Tan2
1Wuxi Medical School, Jiangnan University, Wuxi, China; 2Jiangnan University
Mesenchymal stromal cells (MSC) are an important constituent of bone marrow microenvironment (so called niche) where they support hematopoiesis via direct cell-cell interactions with hematopoietic stem- and progenitor cells, and by releasing soluble factors. Glycans, such as N-glycans, are involved in numerous biological processes, including inflammation, cell–cell interactions, morphogenesis, and cancer development and progression. Recent information provided by lectin-based microarray has revealed novel opportunities to explore the aberrant expressed N-gylcans and their functions in bone marrow niche. Using stromal/hematopoietic in vitro cell coculture system, we firstly investigated the effects of stroma-derived signals on apoptosis sensitivity in co-cultured hematopoietic cells under hypoxia condition. Our results showed that hematopoietic cells KG1a changed its resistance to apoptosis into sensitive to p53-dependent apoptosis after co-cultured with the human stromal cell line HS27a(derived from normal marrow) in hypoxia (1% O2 ). We next applied an strategy using lectin-based glycomic techniques for global profiling of N-glycans in co-cultured hematopoietic cells with bone marrow derived stromal cells under hypoxia. Specifically, we found the enhanced levels of core-fucosylated N-glycans(catalyzed by FUT8) and terminal GalNAc(catalyzed by MGAT3) and elevated levels of their responded genes. Our understanding of the bone marrow microenvironment will be improved by further studies of core-fucosylated N-glycans and terminal GalNAc.
Troy Lund1; Ashley Kramer2; Michael Lehrke3
1University of Minnesota; 2Washington University in St. Louis; 3Mayo Graduate School
The hematopoietic stem cell niche serves to maintain hematopoiesis and recruit cells after hematopoietic cell transplant. Cells comprising the bone marrow niche include: mesenchymal stromal cells, CXCL12-abundant reticular cells, perivascular cells, bone marrow endothelium, adipocytes, chondrocytes, and osteogenic cells. The determinants of homing and engraftment after transplant are incompletely understood, but are mediated by chemokines secreted by niche cells. We determined the transcriptome of both zebrafish and mouse niche cells and found several small candidate proteins that were induced by pre-conditioning radiation. Specifically, dermatopontin (DPT), a 24kDa secreted extracellular matrix protein, was highly expressed in adipocytes (10-fold versus endothelial cells and 7.5-fold versus osteocytes, ANOVA p = 0.02). Immunohistochemistry (IHC) and ELISA confirmed that DPT expression was predominately localized to adipocyte-rich regions of the marrow. After radiation, DPT transcript levels increased 20-fold in the bone marrow (p < 0.01). Homing assays in DPT pre-treated irradiated mice showed a 90% reduction (n = 12, p = 0.02) and a 40% reduction (n = 6, p < 0.05) in whole marrow and lin-sca1+kit+ (LSK) cells that migrated to the marrow 20 hours post-transplant, respectively. This reduction in homing translated to decreases in overall engraftment in primary and secondary transplant recipients.
The bone marrow microenvironment is known to be heterogeneous in its distribution of niche cells. For example, the murine tibia has an adipocyte rich distal end and adipocyte poor proximal end, with the former recruiting 2-fold fewer donor hematopoietic cells versus adipocyte poor regions at 20 hours after adoptive transfer in irradiated recipients (n = 9, p < 0.05). Colony forming unit and apoptosis assays of LSK cells revealed no adverse effects of DPT on hematopoietic progenitor cells, suggesting a specific defect in cell migration as opposed to stem cell toxicity. In vitro adhesion assays demonstrated a 25% decrease in the number of hematopoietic cells that adhered to DPT coated plates (n=6, p = 0.01), and IHC revealed that DPT binds to the endothelial lumen of the vasculature after myeloablative conditioning. CFU assays of circulating cells 20 hours after adoptive transfer showed that DPT led to an increased number of circulating donor derived CFU (149 v 83 CFU/ml, p=0.002), indicating that transplanted cells remained in circulation instead of efficiently homing to the marrow. These data suggest that DPT retards the ability of hematopoietic cells to adhere and transmigrate across the endothelial barrier during the homing process particularly in regions of increase adiposity making DPT a new and novel regulator of hematopoietic cell trafficking.
Samanta Mariani1; Zhuan Li1; Chris Vink1; Elaine Dzierzak2
1The University of Edinburgh; 2The University of Edinburgh and Erasmus MC
Hematopoietic stem cells (HSC) are responsible for the life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs arise in the mouse embryonic aorta-gonad-mesonephros (AGM) region at E10.5 but very little is known concerning the microenvironment that supports their generation. Interestingly, macrophages (Mφ) can be found in the embryo before the production of the first HSC, leading to the hypothesis that yolk sac-derived cells could be involved in the HSC niche. Recent studies in zebrafish revealed that Mφ have a role in hematopoiesis by affecting the migration of hematopoietic stem and progenitor cells from the AGM to the caudal hematopoietic tissue. To investigate whether this mechanism is conserved in mice, we took advantage of the MacGreen transgenic mouse model that expresses EGFP under the control of the Csf1r (colony stimulating factor 1 receptor) promoter. Multi color flow cytometry confirmed that all the EGFP+ cells in the embryo (E8-E11) can be phenotypically defined as Mφ (CD45+CD11b+F4/80+MerTK+CD16/32+Ly6C-Ly6G-). Mφ numbers in the AGM increase over time, with a peak between E10 and E10.5 just before the production of the first HSC. The recruitment of Mφ to the AGM may be due to chemokine receptors/ligands expression by these cells. We found that AGM Mφ are positive for CCR2, CCR3, CCR5, CXCR4 and CX3CR1 by flow cytometry. Moreover, semi-quantitative real time PCRs on FACS sorted AGM endothelial cells from both Macgreen and Ly6AGFP mice, showed CX3CL1, CXCL12 and CCL3expression, suggesting that these chemokines can mediate Mφ recruitment to the aorta. Time lapse vital imaging on MacGreen AGM sections (E10-E11) showed that embryonic Mφ are highly motile cells that undergo mitosis, intravasate into the aorta and interact with CD31+cKit+ cells. Moreover, AGM explant experiments revealed a 40% decrease in the number of CFU-GEMM colonies upon treatment with clodronate liposomes that selectively ablate the 80% of Mφ in the AGM.Together, these results suggest that Mφ play a role in regulating the definitive hematopoietic wave in the mouse and future experiments will focus on specific molecular mechanisms so as to further our understanding on the role that extrinsic factors play in the establishment of definitive hematopoiesis.
Guey Chuen Perng1; Yi-Hui Hsu1; Jih-Jin Tsai2; Po-Lin Chen1; Tzu-Chuan Ho1; Ya-Ping Chen1;Tsai-Yun Chen1; Yu-Chih Lo1; Shang-Rung Wu1
1National Cheng Kung University; 2Kaohsiung Medical University Hospita
Bone marrow suppression resulting in defective hematopoiesis is a known clinical finding in dengue patients. Viral infections may induce damage and/or dampen the potency and capacity of their proliferated and differentiated functions, contributing to imbalance blood components in circulation and to disease development. Yet the physical interactions between dengue virus (DENV) and stem and progenitor cells remain in the shroud of mystery. Experimental designs with multicolor flow cytometry analysis were initiated to document the infection of stem and progenitor cells by DENV. Kinetic results from whole bone marrow cells, isolated stem and progenitor cells, and immune thrombocytopenic purpura donors revealed the importance of stem and progenitor cells in human bone marrow (HBM) for DENV infection. Peripheral blood mononuclear cells (PBMC) from healthy donors also demonstrated that the levels of stem and progenitor cells in the PBMC were a critical component for the DENV infection. Furthermore, results from PBMC of acute dengue patients showed that the levels of stem and progenitor cells were significantly and positively correlated to plasma viral load. Importantly, homing marker investigations on the stem and progenitor cells from PBMC of acute dengue patients revealed that the preferential organs for these cells homing to are gut, regional lymph node, lung, and bone marrow in respective order, suggesting clinical and pathogenic relevant in dengue patients. The cumulative results suggest that DENV infection of stem and progenitor cells in HBM not only lead to destruction of these progenitor cells but also may tailor these cells to serve as viral reservoirs for further dissemination of DENV into distant anatomic sites.
Smrithi Rajendiran1; Scott Boyer2; Anna Beaudin3; Jessica Perez-Cunningham3; Stephanie Smith-Berdan3; Mark Landon3; Camilla Forsberg3
1SOE, UCSC, Santa Cruz, United States; 2Dako, an Agilent Technologies Company; 3UCSC
The lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations are currently unclear. To gain new insights on these questions, we performed quantitative analyses of mature cells produced from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Although our donor chimerism results supported the previous observations for the lineage potential of the various stem and progenitor populations, assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid bias. This bias was accompanied by strong platelet production that far exceeded generation of myeloid, B and T cells, even in the myeloid competent progenitors. Clonal analysis by single cell transplantation and spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. The erythroid-dominant cell production observed by peripheral blood analysis was also evident within individual spleen colonies. Splenectomy differentially reduced erythro- and megakaryopoiesis, but not GM, B and T cell production. However, MegE production was not dependent on the spleen environment, as multilineage reconstitution from transplanted cells was also observed in splenectomized mice. Together, our quantitative comparative assessments of multilineage potential prompt an erythroid-biased model of hematopoietic differentiation.
Maile RomeroWolf1; Maria Quiloan2; Mary Yui2; Jonas Ungerback2; Ellen Rothenberg2
1Caltech, Pasadena, United States; 2Caltech
In order for precursor T cells to progress to a committed state, they must first shut off stem and progenitor programs, and deny access to alternative hematopoietic lineages. Transcription factor Bcl11b is required for T commitment. It comes on sharply at T cell commitment and stays on for the life of the cell. We know that if Bcl11b is deleted before commitment the cells still have access to alternative lineages including myeloid and NK. Our project describes the nature of the changes Bcl11b makes in the gene regulatory network that transition T cells from a progenitor to a committed T cell. Can a "committed" T cell return to a multipotent stem state if a key commitment transcription factor is deleted? Transcription factors like Spi1 (PU.1) that are expressed up until commitment appear to preserve the option of the progenitor cell to become myeloid under permissive conditions. Other genes, like Nfil3 and Zbtb16, enable cells to become natural killers or innate-like lymphocytes. We used Bcl11b conditional knock-out cells to determine whether removal of Bcl11b after commitment can restore access to these alternative cell fates. In vivo, Bcl11b knock out was accomplished using mice that have Bcl11b flanked by lox-P sites and expressing a Cre recombinase driven by a T cell promoter active immediately after commitment (Lck). These Bcl11b KO cells resemble neither pre-commitment nor post-commitment controls. Based on RNAseq analysis from purified from KO cells, some progenitor genes like Kit come back on, but the key myeloid gene Spi1 (PU.1) remains silenced. Others, including Nfil3 andZbtb16, show much higher expression levels in the Bcl11b knock out cells than in wild type cells. When these KO cells are cultured in permissive conditions with or without Notch signaling, some cells divert to NK fate, as indicated by NK1.1 and Dx5 expression by flow analysis. Additionally, the mutant cells without Notch signaling can express markers for alternative cell types including CD11b+Gr1+ putative granulocytes as well asCD11b+CD11c+ putative myeloid dendritic cells. Understanding the action of Bcl11b in this highly defined system helps illuminate how alternative cell fate potentials are extinguished and allows insight into the developing T-cell's gene regulatory network at commitment.
Tamar Saralidze1; Nino Gogokhia2; Tinatin Shvelidze3; Tatia Swanidze4; Tinatin Saralidze5; Lia Mokhevishvili6
1Asiistant Professor, Georgia; 2Tbilisi State Medical University First University Hospital;3Medical Center "IUNONA"; 4Tbilisi State Medical University; 5AIETY Medical School; 6Children's Hospital
Though modern WHO classification of lymphomas are based on the morphological, immunophenotypic and cytogenetic investigations, timely diagnosis and estimation of the degree of malignancy often remains to be a challenging problem that concerns follicular lymphoma (FL) and its atypical forms as well. 30-40 percent of patients with FL eventually transform to more aggressive form and needs high-dose chemotherapy (www.lymphoma.org). Timely diagnosis and prediction of this transformation is also very important. Considering above mentioned problems we searched vein blood leukocyte cultures of patients with lymphoma or suspected on lymphoma according to method worked out by us. This method supports proliferation of malignant cells and reveals proliferative clone responsible for the onset and course of disease [www.e-bookland.ge/ATLAS-of-HEMATOLOGY]. Our research showed lymphocyte proliferation with their atypical forms and lymphocytosis over 3 weeks of cultivation in cases of lymphoma whereas in healthy donors active proliferation of macrophages was observed; We investigated 8 patients with FL. Their lymphocytes were positive for B-cell markers CD10, CD19, CD22, and CD20. In blood leukocyte cultures of these patients atypical lymphoid cells with cleaved nuclei and figures of atypical mitosis were revealed. In vitro FL cells are more cleaved and misshaped than in blood or in lymph node smears that supports to confirm diagnosis especially in cases when due to little amount of lymphoma cells in vivo immunophenotyping is not helpful. In cases of high grade FL abundance of atypical lymphocytes with cleaved nuclei and multiple figures of atypical mitosis in cultures shows high speed of tumor growth or progression and necessity of appropriate treatment. On contrary single atypical lymphocytes with cleaved nuclei in vitro points to low grade FL and indolent course of disease that helps physician to avoid a patient from high-dose chemotherapy and extend "watchful waiting" period. According to our investigations leukocyte culture method can be successfully used for the diagnosis of obscure cases of FL and estimation the degree of malignancy. Culture data not only represents the morphological grade of FL, but reveals tumor progression and predicts clinical course of disease that can help to determine appropriate treatment. Immunophenotyping of in vitro proliferating cells can be held as well.
Mary Saunders1; Pratibha Singh2; Khalid Mohammad2; Melissa Kacena2; Christie Orschell2; Louis Pelus2
1Indiana University School of Medicine, Indianapolis, United States; 2Indiana University School of Medicine
DNA repair and damage responses are critical to maintain and restore genomic integrity following exposure to a variety of exogenous factors. Within the bone marrow niche, normal hematopoietic stem cells are relatively quiescent. Recent evidence suggests that megakaryocytes can promote HSC proliferation and differentiation following injury. Cells of the developing megakaryocyte lineage must remain viable, progress through the cell cycle, and undergo extensive remodeling to maintain mesenchymal stromal and endothelial cells of the bone marrow vasculature. Preserving homeostasis and cellular remodeling of the bone marrow niche requires activation of autophagic pathways. We have found that treatment of mice with non-steroidal anti-inflammatory drugs (NSAIDs) following irradiation promotes autophagy in megakaryocytes in young and old C57BL/6 mice. Following 650 cGy TBI, total marrow CD41+ megakaryocytes and CD71+ proliferating megakaryocytes increase significantly in comparison to non-irradiated control mice. The radiation induced megakaryocyte increase is ablated by NSAID treatment which blocks endogenous PGE2 production. However, the number of SLAM LSK and LSK hematopoietic stem cells is increased significantly in comparison to irradiated control and dimethyl-PGE2 treated mice. NSAIDS also promoted autophagy in normal, non-irradiated megakaryocyte lineage cells. In contrast, in an irradiated megakaryoblastic leukemia cell line, Meg-01, selective inhibition of cyclooxygenase pathways by treatment with meloxicam did not result in the loss of autophagy nor suppression of megakaryopoiesis and differentiation. Interestingly, there was greater than 2-fold increase in the number of megakaryoblast cells and 4-fold increase in thrombocytes. This study represents the first in kind of non-steroidal anti-inflammatory drugs influencing autophagy in megakaryocytes and may confer a protective advantage to cells of the hematopoietic niche following radiation induced injuries.
Stephanie Smith-Berdan; Mark Landon; Leah Kramer; Thessaly Alexander; Camilla Forsberg
Despite the use of hematopoietic stem cells (HSCs) in clinical therapy for over half a century, the mechanisms regulating HSC trafficking, engraftment and life-long persistence after transplantation are unclear. Findings over the past several years have demonstrated that life-long HSC function within bone marrow (BM) niches depends on cells associated with the BM vasculature. We recently showed that the vascular endothelium not only regulates HSC maintenance within BM niches, but also trafficking between the vasculature and BM space. Surprisingly, instead of acting as barriers to cellular entry, we found that vascular endothelial cells, via the guidance molecule ROBO4, actively promote HSC translocation across vessel walls into the BM space. In contrast, we found that the vasculature inhibits the reverse process, as induced vascular permeability led to a rapid increase in HSCs in the blood stream. Thus, the vascular endothelium reinforces HSC localization to BM niches both by promoting HSC extravasation from blood-to-BM and by forming vascular barriers that prevent BM-to-blood escape. Well-established modulators of vascular permeability, such as vascular endothelial growth factor (VEGF) and histamine, had profound effects on HSC location, revealing that vascular integrity is an important regulator of HSC location. In addition, we show that modulation of Robo4 via its putative Slit ligands affects HSC trafficking. Our work aims to uncouple the complex molecular and cellular mechanisms regulating the directionality of HSC migration across vessel walls. These insights will further enhance our ability to regulate HSC trafficking and thereby improve the safety and efficiency of hematopoietic transplantation therapies.
David Stachura1; Julian Aggio2; Anja Brown2; Francis Wright2; Gabriel Marquez2; Clyde Campbell3; David Traver3
1California State University, Chico, Chico, United States; 2California State University, Chico; 3University of California, San Diego
Zebrafish are an excellent model system for studying hematopoiesis due to their external development, high fecundity, and optical transparency. They are also genetically amenable, allowing large-scale mutagenesis and chemical screens for elucidating genes and molecular pathways involved in hematopoiesis to be easily performed. While forward genetic screens have been utilized to identify genes essential for the generation of primitive blood and the emergence of hematopoietic stem cells (HSCs), they have not elucidated genes essential for hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation, due to a lack of methodologies to functionally assess HSPCs. We previously pioneered techniques to test the developmental potential of HSPCs by culturing them on zebrafish kidney stromal (ZKS) cells, a cell line derived from the main site of hematopoiesis in zebrafish. We have also derived zebrafish embryonic stromal trunk (ZEST) cells, isolated from tissue surrounding the embryonic dorsal aorta, the site of HSC emergence in developing fish. In addition to these cell lines, we now report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hour post fertilization (hpf) caudal hematopoietic tissue, the site of embryonic HSC expansion. CHEST cells expand two-fold more hematopoietic cells than ZKS or ZEST cells in vitro, and morphological and qRT-PCR analysis of these cultures indicated lymphoid, myeloid, and erythroid differentiation. Transcriptional comparison of ZKS, ZEST, and CHEST cells will allow the discovery of molecular pathways expressed by these cell lines that support HSPC proliferation and differentiation, but are temporally and ontologically distinct. We have performed RNA-sequencing on these cell lines, and these data allow for the first time a comparison of molecular signals critical for HSPC proliferation and differentiation in the zebrafish, and comparison with mammalian-supportive cell lines should further an evolutionary understanding of vertebrate hematopoiesis. Importantly, they allow the testing and validation of bona fide HSPCs in zebrafish, cells commonly dysregulated in hematologic diseases.
Bilyana Stoilova1; Dimitris Karamitros2; Zahra Aboukhalil2; Georg Otto2; Andreas Reinisch3;Fiona Hamey4; Eshita Sharma5; Marina Samitsch2; Lynn Quek2; Nicolas Goardon2; Bertie Gottgens4; Ravindra Majeti3; Paresh Vyas2
1Weatherall Institute of Molecular Medicine, University of Oxford, UK, United Kingdom; 2Weatherall Institute of Molecular Medicine, University of Oxford, UK; 3Stanford Institute for Stem Cell Biology and Regenerative Medicine, USA; 4Cambridge Institute for Medical Research, UK; 5Wellcome Trust Centre for Human Genetics, University of Oxford, UK
In steady state murine haemopoiesis most blood cells are generated from progenitors downstream of haemopoietic stem cells. However, early progenitor populations remain incompletely defined. We and others have described three human early lympho-myeloid progenitors: lymphoid-primed multipotent progenitor (LMPP), multi-lymphoid progenitor (MLP) and granulocyte-macrophage progenitor (GMP), but the relationship between these progenitors is unclear. Defining early progenitors is not only important to understand normal haemopoiesis but is relevant to our appreciation of differentiation block in human Acute Myeloid Leukemia (AML).
Here, we focus on the earliest human lympho-myeloid progenitors LMPP, MLP and GMP, and using functional (in vivo transplantation, colony assays, quantitative single cell and limit dilution in vitro liquid culture) and transcriptional assays (bulk and single cell RNA analysis) we show: 1. In vitro liquid culture analysis of 1760 single cells and quantitative limit dilution studies showed that LMPP and GMP are highly clonogenic (55-65%) and heterogeneous. In single cell analysis in positive wells, in most cases both progenitors only gave rise to uni-lineage output, with LMPP being lymphoid-biased and GMP myeloid-biased. MLP had poor cloning efficiency of 10% and was exclusively a lymphoid progenitor. Only 10% of the LMPP and GMP had combined lympho-myeloid potential, whereas MLP was not multi-potential in vitro. 2. Transplantation of LMPP and GMP into humanized ossicles in NSG mice gave 1-3% engraftment per 1000 transplanted human cells. The LMPP gave robust GM and B cell engraftment whereas the GMP gave principally GM engraftment. The MLP engrafted poorly with mainly B cell output. 3. Analysis of the expression of 96 lineage-affiliated genes in 900 single LMPPs, GMPs and MLPs showed that the functional heterogeneity was mirrored transcriptionally at the single cell level and that differentiation probably occurs as a continuum. 4. Comparison of the transcriptional programs of normal progenitors and human progenitor-like AML stem cells shed light on the nature of the differentiation block in AML.These data suggest a new model of lympho-myeloid progenitor specification altering our understanding of the molecular mechanisms regulating normal progenitor cell fate choice during haemopoiesis and provide a base for understanding the differentiation arrest in human AML.
Enrico Velardi1; Jennifer Tsai2; Jarrod Dudakov3; Marcel Van Den Brink2; Kimon Argyropoulos2; Malcolm Moore2; Shieh Jae-Hung2
1Memorial Sloan Kettering Cancer Center, New York, United States; 2Memorial Sloan Kettering Cancer Center; 3Fred Hutchinson Cancer Research Center
A tightly regulated network of intrinsic and extrinsic signaling pathways exists to preserve hematopoietic stem cell (HSC) pool size and function. This is particularly relevant during periods of hematopoietic stress or injury when HSCs are induced to exit their quiescent state and transiently enter into a program of proliferation and differentiation to replenish blood loss.
There has been growing interest in how long-range effects of circulating sex steroids integrate HSC function with overall tissue physiology. Here we describe a novel and unexpected role of luteinizing hormone (LH) in regulating HSC biology independently of downstream sex steroids. Our findings revealed that the most primitive human and murine HSCs highly expressed the LH receptor. LH administration stimulated HSC proliferation whereas LH ablation promoted HSC quiescence after hematopoietic insults, including radiation exposure, Poly I:C treatment and repeated rounds of chemotherapy.
Uncontrolled proliferation after hematopoietic injury, such as chemotherapy or irradiation, has been associated with HSC exhaustion, long-term myelosuppression and ultimately death. We found that pharmacological inhibition of LH using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) 24h after a lethal dose of irradiation (L-TBI) promoted hematopoietic recovery and mouse survival. We could demonstrated that the regenerative effects were independent from downstream sex steroids given that administration of LHRH-Ant improved survival rates in surgically castrated mice following radiation injury, while surgical castration alone did not. To definitively determine whether the regenerative effects of LHRH-Ant treatment depended on suppression of LH, we administered the LH receptor agonist human chorionic gonadotropin (hCG) to LHRH-Ant treated mice that had been given L-TBI one-day prior. Consistent with our hypothesis, administration of hCG abrogated the beneficial effects of LHRH-Ant on survival after radiation injury. Taken together these data not only reveal an unexpected role of LH in regulating HSC homeostasis, but also shows that inhibition of LH signaling using a single dose of an LHRH-Ant represents a rational and feasible approach to preserve the HSC pool, after high dose radiation, thereby mitigating acute hematopoietic radiation syndrome.
Tienan Wang1; Yi Wu2
1Hong Kong University of Science and Technology, Hong Kong; 2Hong Kong University of Science and Technology, China
Microglia are central nervous system (CNS)-resident macrophages and play important roles in neural development and function. Yet, how microglial precursors born in peripheral hematopoietic organs colonize the CNS remains undefined. Here, by utilizing in vivoimaging and genetic manipulation of zebrafish, we showed that microglial precursors enter the optic tectum of the midbrain, where the majority of microglia reside during early development, via the lateral periphery between the eyes and brain and the ventral periphery of the brain in a circulation-independent manner. Intriguingly, long-term colonization of the optic tectum by microglial precursors is driven by apoptotic neuronal death in the midbrain and involves homing and settling processes. We further provide evidences indicating that the homing process is mediated in part by lysophosphatidylcholine (LPC), a phospholipid known to be released from apoptotic cells. Our study reveals that microglia colonization of the developing midbrain is triggered by neuronal death naturally occurred during neurogenesis possibly via releasing chemoattractant.
Tao Yu1; Ye Tian2
1Hong Kong University of Science and Technology; 2Hong Kong University of Science and Technology, China
Microglia are central nervous system (CNS)-resident macrophages and they play pivotal roles in the CNS development and homeostasis.Recent fate mapping studies have revealed that zebrafish has two different microglia populations, which arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA) and manifest distinctive colonization kinetics and potential. Yet, the genetic programs governing their development remain largely undefined. Here we showed that the development of the RBI- and VDA-derived microglia is orchestrated by distinctive regulatory networks, vertical and parallel, formed by the Ets transcription factors Pu.1 and Spila, respectively. Epistatic studies further revealed that the two distinctive regulatory networks control the development of microglia derived from the RBI and VDA largely through regulating a common downstream regulator Irf8. Our study indicates that tissue-specific transcription factors Pu.1, Spila and Irf8 form distinct genetic networks to control the development of microglia of different origins.
Claudia Waskow1; Gulce Percin2
1Regeneration in Hematopoiesis, TUD, Germany; 2TUD
Dendritic cells (DCs) are potent antigen presenting cells that depend on Flk2-mediated signals for their differentiation. In Flk2-deficient mice, however, a small but functionally active fraction of DCs remains. We show here that the combined deficiency for Flk2 and Csf1r, another member of the same family of receptor tyrosine kinases, results in natural DC null mice. In contrast to Flk2, Csf1r-mediated signals affect DC differentiation by a cell extrinsic, non-hematopoietic mechanism that does not require Csf1r signaling within the entire adult mouse. During embryonic development Csf1r signaling is crucial for the generation of macrophages, which emerge from erythro-myeloid progenitors in the yolk sac before the presence of definitive hematopoietic stem cells (dHSCs). The tissue-resident red pulp macrophages (RPMp) in the spleen are such embryonic macrophages, and we show here that Csf1r signaling is required for their differentiation. In the absence of Flk2-mediated signals, loss of Csf1r expression results in complete ablation of spleen DCs in vivo due to the lack of embryo-derived RPMps. The dependency of a normal DC pool size on the presence of tissue-resident macrophages maintains throughout adulthood of the mice, suggesting that there is a continuous cross-talk between myeloid cells of adult and embryonic origin, and this communication is a crucial regulator for hematopoietic cell pool size control in vivo. RPMps have been implicated in the maintenance of tissue hemostasis, removal of pathogens and clearance of cellular debris. We assign here a novel important role to RPMps in the maintenance of tissue homeostasis by regulating the pool size of dHSC-derived myeloid cells in the spleen.
Joan Yuan1; Trine Kristiansen2; Elin Jaensson Gyllenbäck1; Alya Zriwil2; Tomas Björklund2;Jeremy Daniel3; Ewa Sitnicka1; Shamit Soneji1; David Bryder1
1Lund University, Lund, Sweden; 2Lund University; 3Copenhagen University Center for protein research, Copenhagen, Denmark
Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigate whether the well-established developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. In vivo single cell fate mapping was achieved by cellular barcoding to resolve developmental changes in lineage potential. We demonstrate that fetal liver definitive HSCs give rise to both B-1a and B-2 cells at the single cell level. Further, B-1a potential is selectively diminished in all HSCs with time while B-2 output is maintained. Importantly, B-1a / B-2 plasticity can be reinitiated in a subset of adult HSCs by ectopic expression of the fetal RNA regulator Lin28b. Using barcodes as unique cellular identifiers, we establish a direct link between Lin28b induced B-1a plasticity and fetal-like HSC behavior including elevated HSC self-renewal and repopulation potential.
Alya Zriwil1; Charlotta Boiers2; Lilian Wittmann1; Joanna Green3; Petter Woll4; Sten Eirik Jacobsen4; Ewa Sitnicka1
1Lund University, Lund, Sweden; 2Lund University, Sweden; 3University of Oxford, Oxford, UK; 4Karolinska Institutet and Center for Hematology and Regenerative Medicine, Oxford, U.K.
While it is well established that unique B cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the Colony Stimulating Factor-1 receptor (CSF1R), previously thought to be expressed and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes, and in line with this possess residual myeloid as well as B cell, but not T cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.
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Native Plants
Search for native plants by scientific name, common name or family. If you are not sure what you are looking for, try the Combination Search or our Recommended Species lists.
Solidago hispida
Solidago hispida Muhl. ex Willd.
Hairy Goldenrod
Asteraceae (Aster Family)
USDA Symbol: SOHI
USDA Native Status: L48 (N), CAN (N)
Similar to Solidago bicolor but with yellow flowers instead of white. Solidago hispida is found from New England south to Georgia, west to Arkansas, and north to Illinois, Minnesota, and much of the eastern two-thirds of Canada.
From the Image Gallery
Bloom InformationBloom Color: Yellow
Bloom Time: Aug , Sep , Oct
DistributionUSA: AL , AR , CT , DC , DE , GA , IA , IL , IN , KY , LA , MA , MD , ME , MI , MN , MO , MS , NC , NH , NJ , NY , OH , OK , PA , RI , SC , SD , TN , VA , VT , WI , WV
Canada: MB , NB , NL , NS , ON , PE , QC , SK
Growing ConditionsSoil pH: Alkaline (pH>7.2)
CaCO3 Tolerance: High
BenefitConspicuous Flowers: yes
Value to Beneficial InsectsSpecial Value to Native Bees
Special Value to Honey Bees
Supports Conservation Biological Control
This information was provided by the Pollinator Program at The Xerces Society for Invertebrate Conservation.
Additional resourcesUSDA: Find Solidago hispida in USDA Plants
FNA: Find Solidago hispida in the Flora of North America (if available)
Google: Search Google for Solidago hispida
MetadataRecord Modified: 2013-06-15
Research By: TWC Staff

subset: commoncrawl


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Dive Site: Perry Street Rock Pile | Perry St. Rocks MAP IT!
City: Hollywood, Florida
Type of Dive: South Florida Beach Dive
Depth: 12-20 Feet
Parking: Metered | Pay at the master meter with cash or credit card, $4.35/hr
Last Updated: 6/2/2019
The Perry Street Rock Pile | Perry St. Rocks Beach Dive:
The Perry Street Rock Pile is an artificial reef that runs north-south from Palm Street to Allen Street. Huge boulders the size of cars make up the Perry Street Rock Pile. This South Florida dive location is similar to Turtle Reef or South Inlet Park, but the boulders here are MUCH bigger. Unlike other reefs, at the Perry Street Rock Pile you can swim between the rocks! This makes the Perry Street Rock Pile a fun and engaging dive site.
The Perry Street Rock Pile starts 600 feet off the beach and is about 150 feet wide (east-west). The average depth here is 12-20 feet (some of the boulders have high peaks).
This project was known as Beach Renourishment Project, Segment III by Broward County. Completed in 2006, it is now an amazing reef for scuba divers to enjoy!
Like most of South Florida, you MUST use a dive flag when diving at the Perry Street Rock Pile.
Driving Directions to the Perry Street Rock Pile In Hollywood:
From I-95, take exit 23 for Griffin Rd. and head east. Turn right onto Federal Highway / US1. Turn left onto Dania Beach Blvd. Follow this road and watch out for the fork in the road. Keep left at the fork, follow the signs for Florida A1A south. Follow the loop that will bring you onto A1A south / Ocean Dr. Turn left onto Perry Street. This is where you want to park for the Perry Street Rock Pile.
What You May See At The Perry Street Rock Pile:
French Angelfish, Queen Angelfish, Squirrel Fish, Porcupinefish, Sergeant Majors, Yellowtail Snapper, Parrotfish, Grunts, Hogfish, Porkfish, Nurse Sharks, Southern Stingrays
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Write your own review by leaving a reply on the bottom of this page!
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Submit your photos to firstname.lastname@example.com and we'll make sure you receive proper credit!


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Nelsonville Coal Mine 1908
Posted by JRW on February 17, 2011
4 Responses to "Nelsonville Coal Mine 1908"
June 25, 2012 at 6:14 pm
Does any one know the location of this mine?
February 8, 2014 at 7:14 pm
The page heading mentions Nelsonville. Do you know that it's in Nelsonville and are looking for a specific hill?
February 26, 2014 at 8:57 am
Yes. I am looking for a specific location. There used to be a coal tipple that loaded canal boats or trains across from my house in Nelsonville. This sort of looks like the hill but I am not sure. I was just curious. Thanks for the response.
- February 26, 2014 at 8:57 am
- February 8, 2014 at 7:14 pm
February 23, 2014 at 10:39 pm
It was near Hocking College behind the Nature Center.


text: logging in or signing up Stars and the solar system TheChamp2 Download Post to : URL : Related Presentations : Let's Connect Share Add to Flag Embed Email Add to Channel Copy embed code: Embed: Flash iPad Dynamic Copy Does not support media & animations Automatically changes to Flash or non-Flash embed WordPress Embed Customize Embed Use HTTPS URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 8782 Category: Science & Tech.. License: All Rights Reserved Like it (1) Dislike it (1) Added: February 10, 2012 This Presentation is Public Favorites: 3 Presentation Description This the small presentation on Class 8th Chapter: Stars and the solar system. Comments Posting comment... Premium member Presentation Transcript PowerPoint Presentation: Stars, planets, the moon and other bodies in the sky are called celestial bodies. The different shapes of the disc of the moon observed during a month are called the phases of the moon. Nearly fifteen days after the new moon can be seen approximately once a month. The new moon is seen when the moon is in between the sun and the earth. The full moon can be seen when the sun and the moon are on either side of the earth. The surface of the moon is dusty and full of craters. The moon has a very thin atmosphere. This is why sound transmission is not possible on the moon. The moon has a very thin atmosphere. This is why sound transmission is not possible on the moon. A Light year is used to measure very large distances. It equals the distance travelled by light in one year. Sunlight takes about 8 light minutes to reach the earth.PowerPoint Presentation: There are 8 planets in the solar system that revolve around the sun. The sun is a major source of heat and light for all the planets in the solar system. Planets reflect the Sunlight that is incident on them. They have no light of their own so they don't tinkle like stars. Planets have definite paths called orbits in which they revolve around the Sun. The time taken by a planet to complete one full revolution around the Sun is called its period of revolution. The taken by a planet to rotate a full 360 degrees on its axis is called its period of Rotation. A celestial body that revolves around another celestial body is called a satellite.PowerPoint Presentation: Mercury is the smallest planet in the solar system as well as the closest to the Sun. It takes about 88 days to complete one revolution around the Sun. Venus is the second closest planet to the Sun. It takes about 225 days to complete one revolution around the Sun. It has no satellite or moons. It rotates from East to West. Earth is the only planet on which life is known to exist. It takes 365 days for the Earth to complete one revolution around the Sun. Mars completes one revolution around the Sun in about 687 days. It has to Moons of its own. Jupiter is the largest planet in the solar system. Jupiter's four larger Moons are called lo, Europa, Callisto and Ganymede. It rotates the fastest among all planets. The rings of Saturn are made of ice particles and dust. Saturn is the only planet that is lighter than water. The largest of Saturn's Moon is Titan. Uranus is the coldest planet. Neptune as discovered through mathematical calculation.PowerPoint Presentation: Mercury, Venus, Earth and Mars are called the Inner Planets. Jupiter, Saturn, Uranus and Neptune are called the Outer Planets. The Outer Planets have several Moons and a system of rings.PowerPoint Presentation: Apart from the sun and the planets, the solar system also comprises of celestial bodies such as meteors, comets and artificial satellites. A meteor is made of debris. It enters the Earth's atmosphere at a very high speed. The friction with the atmosphere at a very high speed the friction with the atmosphere makes the meteor hot and it burns till it disintegrates. As it falls to the ground, it glows brightly. This is why it is called shooting star. Some meteors reach the ground before they burn completely and evaporate. These are called meteorites . Asteroids are made of rubble and are found in the gap between the orbits of Jupiter and Mars. The gap is called asteroid belts. An asteroid does not have a tail. However, a comet is made up of a bright head and a long tail. A Comet is comprised of rock, dust, water, ice frozen gases. Artificial Satellites are man-made and are launched from the Earth. They are used for transmission of television and radio signals, telecommunication and remote sensing.PowerPoint Presentation: Constellation is a group of stars that form a recognizable shape. Ursa Major is in the shape of a bear. The Big Dipper is a group of stars that from part of this constellation, it represents the black half of the bear's torso and its long tail. It can be used to locate the position of the Pole Star. Orion has seven or eight bright stars. The three middle stars make stars make up the belt of Orion. The four other stars are in form of a quadrilateral. Cassiopeia resembles a distorted W or M. Stars appear to move from east to west through the night because the Earth rotates from west to east. You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.


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Happy Earth Day! I hope to spend today, ideally, puttering about my garden. Yes, we can all aspire to do something more high-minded (and Barry Dennis, sitting right below me in the blog rail, will tell you how), but even just beautifying your own patch of soil contributes to the pleasure of everyone around you (including even something as simple as a window box if you have no outdoor earth to plant).
Relaxing, too, will help me recover from what was truly an amazing week here at HuffPost -- and what promises to be another in the coming week. First off, in home news, Immigration Minister Jason Kenney joined our editorial team on Wednesday for dinner -- and a no-holds-barred question session, including three questions selected from our readers. Kenney gamely and straightforwardly answered everything put to him -- from issues ranging from the recent and controversial immigration reform measures he has proposed, to what he would do to solve the status of "Invisible Canadians." You can also view a slideshow and video of Kenney's visit here. In the end, the minister stayed for two hours -- managing to get a few bites of chicken and salmon in between the questions, before heading off to Toronto's Billy Bishop Airport on an extended cross-country tour to discuss the reforms with Canadians. Give the guy this: He should certainly qualify as a top finalist for our most hard-working and inexhaustible cabinet minister. Afterwards the minister tweeted:
Note to Canada's editorial boards: up your game. @HuffPostCanada does it with style. They make two hours of tough questions seem like fun!
Meanwhile the conservative knife-fight in the Alberta provinicial election continued, with Danielle (great name!) Smith's stepping up to defend two of her more controversial Wildrose candidates over racial and anti-gay comments. Follow our instant coverage of the election results tomorrow as Albertans head to the polls.
More gay controversy took place on the other end of the country -- in this case tragically so, when Nova Scotian gay activist Raymond Taavel was beaten to death by a diagnosed schizophrenic. As the controversy moved to focus on what went wrong in the mental health system that resulted in the public release of a known "risk" patient -- Andre Denny, now charged with murder -- blogger Benjie Nycum urged for the discussion to move beyond the simplicity of "the blame game" by asking what would Taavel do?
We need to get over our "broken system"/"heads should roll" approach to assigning blame in order to solve problems. Raymond Taavel would have approached the problems that led to his cruel, unnecessary, and unfortunate death with sincerity, compassion, urgency, and persistence. The blame game has started, and I think Taavel would agree that it is none of the above.
Also here in Blogtown, Douglas Anthony Cooper posted his next instalment of his astonishing expose of the killing practices of the People for the Ethical Treatment of Animals [PETA]: In this post he applauded Jennifer "Katniss" Lawrence for having the courage to tell the group (after it criticised the actress for skinning a squirrel onscreen): "Screw PETA."
And Cooper's series should cause more skepticism towards PETA's sexy PR antics -- such as when, earlier this week, vegan activists from the group crashed Toronto Mayor Rob Ford's weekly public weigh-in, wearing bikinis made of lettuce leaves. Perhaps Mayor Ford should crash the group's next pet killing session at their headquarters in Norfolk, Virginia? He could carry a sign: "I may eat meat but I don't kill healthy, adoptable puppies and kittens for no good reason." (See Cooper's series, above.) Just please, Mayor Ford, spare us all and don't do it in a bikini.
And maybe schedule your visit there when the Gay Pride parade is taking place, so you can at least have a better excuse for not showing up this year.
Also, read Peter Worthington's fascinating recollections of visiting serial murderer Clifford Olson at the notorious Kingston Penitentiary, which authorities promised to shut down. And in sports, Dave Menzies' widely shared sentiment on why he hates the Canucks.
Debate continued over the 30th birthday of the Charter: Worth it? Or not? Irwin Cotler thinks so, while Steve Stinson says "meh." And Veterans Affairs Minister Steven Blaney took to our blog rails to take issue with another blog, by NDP MP Irene Mathyssen, that accused the government of not doing enough for homeless vets.
O, and did anyone notice the Big O was in the country, offering a "lifeclass" in Toronto under Mountie escort? Our lifestyle team was there, and helpfully reported on what you can learn from Oprah in two hours.
Lastly (before I head out into the garden), may I remind you that the Huffington Post Canada is rapidly becoming the go-to destination for literary output? Our cultural-minded readers will remember that you were able to read in advance excerpts from the finalists for the Charles Taylor Prize for Literary Non-Fiction, awarded in March. Readers were treated this past week to exclusive excerpts from Noah Richler's new book, What We Talk About When We Talk About War. Starting this week, as in tomorrow, we will be excerpting the finalists for both the $50,000 Donner Prize for best public policy book (to be awarded May 1), and the $25,000 Shaughnessy Cohen Prize for Political Writing (to be awarded on Wednesday).
AND -- shhhh, wait for it -- HuffPost has scored the exclusive advance rights to David Frum's much buzzed about new political satire, Patriots, which will debut in this space tomorrow, as well as on the U.S. and UK Huffington Post sites.
It's the first foray into fiction for Frum, who writes (in a blog that will accompany the first chapter tomorrow):
'As some things are too strange for fiction, others are too true for journalism.'
That quip belongs to P.J. O'Rourke. The idea behind the quip explains why I turned to fiction to try to describe what has happened in Washington over these past four years of national crisis.
How can the political elite of a great democracy so utterly abdicate responsibility during an economic depression? Why has extremism flourished and compromise become so unreachable?
Who are the real players -- and what's the game they are playing at?
I could tell you. Or I can show you.
Arianna writes of Patriots: "David has been a friend -- and HuffPost blogger -- for years. He is someone who fearlessly speaks his mind, regardless of where the chips may fall, so it's no surprise he's able to convey so much truth in his fiction. We know our readers will eagerly anticipate engaging with each new chapter as it's revealed on our site."
You can also read Frum on the New York memorial for the late Christopher Hitchens, which occurred Friday. I was there too. I don't think ever in my lifetime have I seen, or will see again, so many extraordinary literary talents and minds gathered together under one roof (in this case, the historic Cooper Union hall) -- from James Fenton, Salman Rushdie, Ian McEwan to Martin Amis and Christopher Buckley (and those were just the speakers. The audience increased the wattage.) For two hours, Christopher lived again: through the words of Christopher's the speakers chose to read, to a slideshow of photographs, and finally, to a wonderful collection of clips. They were there for him, and now he is gone.
Christopher is someone I'll be thinking of out in the garden, this Earth Day, planting things. Yes, we all end up in the earth eventually -- all the more reason to embrace it, and beautify it, and enhance it in every way we can, while we are still lucky enough to live.


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Earlier when I heard of PolyBrowser I thought it would be just another browser with tweaked user interface, but then I installed it in my MacBook Pro which runs Mac OS X 10.10 Yosemite and I found its completely different than other browsers.
The extra features which are present in PolyBrowser are not there in any major browser. Lets first talk about its features then we'll move towards downloading and installation of PolyBrowser.
PolyBrowser Features
- Websites are stitched together: which means you can scroll horizontally between tabs, without actually switching between tabs.
- Zoom In and Out: Suppose you have 5 tabs opened in your PolyBrowser window, you can zoom out to see the larger picture of your all tabs.
- Drag Handles on sides to resize the opened tab: Which means you can see more than one tab at a time without taking out tab to new window. By this feature you can optimally use your screen size.
- Side bar search: You can expand the sidebar and do a google search without opening the new tab.
In short, this browser is perfect for the users who like multitasking on web.
Download & Install PolyBrowser in Mac OS X
PolyBrowser is only available for Mac OS X 10.10 Yosemite, OS X 10.9 Mavericks, OS X 10.8 Mountain Lion and OS X 10.7 Lion.
Step 1: Go to official download page and download PolyBrowser for Mac OS X.
Step 2: Extract the downloaded file and open PolyBrowser-2.0.*dmg file.
Step 3: You might get warning saying "this is application downloaded from internet. Are you sure you want to open it?", ignore that and open it.
You are done with installation now open it and enjoy your multitasking on web. And if you have any issues, please let me know by posting comments.


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The Miller Elder Law Firm, PA
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Gainesville, Florida 32606
Phone: (352) 379-1900
Fax: (352) 379-3926
Shannon M. Miller
Board Certified Elder Law Attorney
Genna Fasullo Lapeer
Elder Law Attorney
Sandi Robertson
Courtney Brown
Korene Clemings
Legal Assistant
Rachel Barr
Office Assistant
Why You Should Contact the Miller Elder Law Firm
In the midst of great distress between dealing with the passing of a loved one, and facing the possibility of estate litigation, it is imperative to immediately contact a proper Elder Law attorney who can assess and respond to the legal issues in question in a timely manner.
The Miller Elder Law Firm provide you with prompt and competent legal counsel that effectively responds to any of the following needs regarding Guardianship, Special Needs Planning, Estate Planning, Medicaid Planning, Probate, Trust Litigation and Exploitation.
Contact The Miller Elder Law Firm today for a free initial consultation*. Call our Gainesville, Florida office at (352) 379-1900 or contact us online (millerelderlawfirm.com).
*Free initial consultations are not provided for Medicaid and Exploitation.


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Furniture Terms – N
nail head trim: the decorative use of a line of tacks at fabric / leather attachment points on upholstered goods.
Naked Leather: Pure Aniline.
nap: a soft or fuzzy surface on fabric or leather.
Nature's Signatures: Naturally occuring characteristics on leather. Bites, stings, wrinkle and cuts.
neoclassical style: Revivals of interest in ancient Greek, Roman, and Egyptian themes, which occurred during the Renaissance, Adam, and Empire eras, and especially in the late 18th century, when appetites for it were whetted by archeological discoveries.
nesting table: concentrically sized tables, usually three, so that the smaller sizes may be tucked under the larger.
night stand: a small table used beside a bed, often incorporating drawers. In recent years the term has become more generalized and used in reference to its function rather than construction.
Nubuk Aniline: Top grain leather that has the "nap" effect cause by removal of the epidermis.


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Gregg Braden
For over 22 years, Gregg Braden has searched high mountain villages, remote monasteries and forgotten texts to uncover their timeless secrets. Combining his discoveries with the best science of today, his original research crosses the traditional boundaries of science, history, and religion offering fresh insights into ancient mysteries. In doing so he has redefined our relationship to our inner and outer worlds, while sharing his life-affirming message of hope and...Read More


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Book Day for Little Libraries and Help The Rural Child!
Shelanti celebrates it's annual Book Day with Help The Rural Child and Little Libraries!
Help The Rural Child!
Little Libraries
"After having visited many creches and daycares in the townships around Cape Town I realised that caregivers do their utmost to enhance the daily lives of the children placed in their care, but 'luxuries' like books and stationery are unattainable although they are key ingredients to creating a stimulating environment.
By launching the little libraries initiative we wanted to provide those creches, daycares and pre-schools with a reading corner where kids can learn, dream and enjoy a whole new world through words and pictures - unlocking their imaginations. That way, children can also become familiar with books prior to arriving at primary school where they will be handed textbooks.
Originally we aimed to help one school. A year later, we had helped 40 - which represents 5000 books, enabling 1500 children to have access to reading material. We want to help many more!!!!
We also started distributing stationery to ensure that children get to develop their fine motor skills and thus do not arrive at primary school not knowing how to hold a pen as yet" Camille, founder
Knysna Fire
In light of the fires that devastated the community of Knysna earlier in 2017, our learners were encouraged to donate items. These items were then driven up to Knysna by the Baker family (Tyla, Grade 3; Daniel, Grade 6) and added to the other donations collected to contribute to helping those who lost everything.
Sidewalk Specials
African Tails
Once upon a time in December 2006, an organisation called African Tails was born. Its is set on the dusty streets of Du Noon and Joe Slovo townships in Cape Town, where African Tails strives to curb the over-population and suffering of abused and neglected township dogs.
Everyone at African Tails is bound by their love for the uniquely African dog, known as Canis Africanis, frequently found in rural areas and on the streets of South Africa's informal settlements.
Fallen Angels
Fallen Angels Pet Rescue is a registered NPO (NPO 175-638) focusing on the rescue, rehabilitation and rehoming of domestic animals.
Any animal that is forgotten, abandoned (abused), lost, lonely, emaciated and neglected (FALLEN) is considered to be a Fallen Angel & therefore part of the focus area of this organisation. Through animal therapy they aim to assist in healing the broken souls of any "FALLEN Angels" they are financially able to assist.


text: I notice this year more than ever, all the major corporations from SAP to Infosys to Kroger are superimposing their corporate logos on to a rainbow flag to show how much they care about LGBTQLMNOP Pride month. Anyone else think that this is much ado over a lifestyle which merely entails misplacing and misuse of their human naughty bits? Why no increased awareness of Hetero Pride?


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Predicting the Ebb and Flow of Auto Glass Repairs in Tucson
In training seminars, they teach the individuals to spot very specific cues and really study an object in incredible detail. People unfamiliar with the certain item will miss the nitty gritty peculiar details that trained professionals can notice. One of these very important areas is in auto glass repair. auto glass repairs in Tucson require a very precise and calculated eye.
Of course, a total and complete crack down the middle is impossible to miss. But when precision is measured, it accounts for two things. One is the quality of the repair itself. Some glass can be salvaged, and professionals can work in the glass in a way that is cost effective but still restores it to a pristine condition. The other way to measure precision is with how the glass is damaged.
It is hard to predict how glass will evolve and crack. For example, a rock will chip a small piece of glass. it makes a noticeable indent, but it looks rather harmless. But over time, that little nick in the window could start spreading into a longer crack. It could then seep into the side of the glass. When it reaches the edge, it has ruined the integrity of the entire glass. If another rock was to hit at any other locations, for the most part, the glass will likely shatter and cause serious damage to the driver and passengers.
On the other hand, a small chip could legitimately be harmless for a long period of time. Will the glass randomly shatter? It is unlikely, and no Auto Glass Repairs in Tucson should tell an individual that this is true. But it is cause for concern. It is even more important to get auto glass repairs if the crack is obstructing the driving field of vision. One may be victim to a driving ticket on top of a broken windshield.
Max Auto Glass in Tucson stresses the importance of repairing a glass with a crack. At the very least, individuals can bring them down and get the situation assessed by a professional. It is hard to predict with 100% accuracy how a chip or crack will evolve. But there are signs to look for, and different glass makes often work in different ways.


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A few days, Nokia had released the PR1.3 firmware update for the N900 which allows the device to dual-boot between Maemo and MeeGo OS. Following this, the MeeGo OS v1.1 for handsets has been released today as well with U-Boot support. No official change-log has been provided so we can't really say what the major changes in this version are. However, we expect everything to be more polished and simply work better.
Since Nokia does not officially support N900 owners dual-booting their phones, I would suggest them to stay away as it will void their warranty. However, hackers and modders who like to tinker around with their phone are already in action and have uploaded a video of the N900 dual-booting the Maemo and MeeGo OS. Here is the video :
Users can download the MeeGo OS v1.1 image for the Nokia N900 from here. Let the modding begin folks!


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The Balancing Act: 6 Time Management Tips for Teens
Ah, time management! That surprisingly elusive talent/asset that frequently feels integral to success. It's a critical skill to have and one that you should certainly try and cultivate. After all, the further you get into high school, the more important it becomes. Indeed, you'll most likely be juggling schoolwork, extracurricular activities, a college search and SAT/ACT prep all while you attempt to maintain a healthy social life. We realize that's a tall order. So, how do you best manage your time?
Push yourself to make a to-do list. To-do lists are a fabulous way to help you stay organized. They'll force you to take a mental count of everything that's going on and offer a physical reminder of what needs to get tackled. You can craft the list so that the most crucial and/or immediate tasks appear first. And you can even decide to sprinkle a few fun activities throughout to keep yourself motivated. Plus, you'll get the satisfaction of crossing off completed jobs and hold tangible evidence of your accomplishments.
Think about when you're most productive. We all operate differently. Some of us are morning people who feel like crawling into bed before the sun even sets. Others are night owls who don't feel fully conscious until at least 3:00 in the afternoon. Certainly, there's no right or wrong answer (though hopefully your optimal time is not 4:00 am). Regardless of when it is, think about tackling your work then. You are likely to be more focused and efficient and retain more information that way.
Make sure you maximize your time. Whether it's a long bus ride home, waiting at the doctor's office or having a half hour to kill between school and soccer practice, you frequently have moments of down time. You should use those instances to your advantage, conquering a few Algebra problems or reading the next assigned chapter in The Great Gatsby. You'll have less to worry about when you get home and it might even allow you to get ahead (or, at the very least, net more evening relaxation time).
Keep a calendar. It doesn't matter if you use your Gmail account, iPhone or a good ol' fashioned desk calendar. You can keep track of class schedules and assignments, daily/weekly chores, upcoming tests, social events, big sports matches, etc. By (regularly) maintaining said calendar, you'll know what to expect for each upcoming day. And you'll easily be able to prepare for what's coming down the proverbial pike.
Figure out how to properly budget your time. Take a moment to think about how long certain assignments and activities typically take. If physics homework usually requires a full hour and jazz band is always two hours, you'll know that you have to allow for that in your daily schedule. Once you solidify/clarify all of your commitments, you'll be able to figure out a weekly agenda. And, importantly, you'll clearly see where you can pencil in some time to kick back and relax.
Realize that it's okay to say no. Unfortunately, there are only a finite number of hours in the day. And sometimes you simply can't accept every invitation that's extended. If you are already attending baseball practice and a study group, you probably shouldn't also agree to go to the movies. You need to recognize your commitments and priorities. A trip to the local cinema can always be rescheduled.
Time management is an essential skill. Certainly, it helps to alleviate stress and anxiety. Perhaps more importantly, it helps to ensure that we're performing to the best of our capabilities. And while some of us might be more naturally inclined to organization than others, it's an attribute we can all develop with guidance and practice.


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Regular price
Regular price
This organic tonic, described as an "unsung hero" by PbAO customers, not only hydrates, tones and revitalizes tired skin, but also sets your makeup, cleanses post-workout, and most notably, shifts your energy—inviting an elevated state of being!
You will receive the healing benefits of Reiki when you use this spirit-lifting tonic. Be it post yoga or pre-meditation, to calm frayed nerves or welcome you to the day. This aromatic heart and third eye chakra opener is a centering compliment anytime. Our Rose Geranium Hydrosol helps soothe pre-menstrual symptoms while Palmarosa purifies your energy, giving you an open frame of mind whenever needed.
To create this buoyant and radiant glow we used Sacred Tonic, Ageless Skin Serum, Healing Salve and Lip Repair.
Sacred Tonic is made with the finest natural, wild-crafted and organic ingredients—by hand in small batches to ensure potency and freshness.
All Ingredients are Organic or Wildcrafted.
Natural Distilled Water, Witch Hazel, Pelargonium graveolens (Rose Geranium) Hydrosol, Aloe barbadensis (Aloe Vera), Vodka, Organic Essential Oils: Cananga odorata (Ylang Ylang), Pelargonium (Geranium), Lavandula angustifolia / lavandula officianalis (Lavender), Cymbopogon martinii (Palmarosa)
*Results are based on individual experiences
1. Spritz your freshly cleansed face with a generous mist, prior to applying Ageless Skin Serum.
2. Atomize again after makeup application.
3. Use it to freshen after a workout, or whenever you need to breathe deeply—to reset your mood and access a higher state of being!
4. Use it right before bed to ease you into your nightly slumber. And share it with your kids to help when they need a little time to move through big feelings.