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The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.

The structure-based design of constrained alpha-helical peptides derived from the viral suppressor of RNA silencing TAV2b is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased alpha-helicity and protease stability. One of these modified peptides (B3) shows high affinity for different double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry.

Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing–based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II–expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors. CA Trilling

The transcription factor RUNX1 is a master regulator of normal hematopoiesis and is involved in cell fate decisions. RUNX1 mutations have been shown to contribute to the development of myeloid neoplasms, and in myelodysplastic syndromes (MDS) it is one of the most frequently mutated genes. Such mutations lead to RUNX1 proteins that lack transactivation activity or DNA-binding ability resulting in a loss of its tumor suppressor function. The dominant-negative short isoform RUNX1a resembles truncated RUNX1 mutants and inhibits the function of the full-length RUNX1 proteins. Additionally, a recently published study identified overexpression of RUNX1a but not full-length RUNX1 in CD34+-cells from patients with myelodysplastic/myeloproliferative disease, which increased with disease progression. This strongly suggests that truncated RUNX1 plays a pivotal role in myelodysplastic disease. However, the precise molecular functions of mutating RUNX1, particularly with respect to the identity of RUNX1 target genes conferring its tumor suppressor function, remain unclear. Previously, our group reported that overexpression of RUNX1a immortalized murine hematopoietic stem and progenitor cells (HSPCs) in vitro. Immunophenotyping of these cells confirmed the expansion of an immature subpopulation defined as Lin- Sca1+ Kit+ (LSK). This phenotype was reversed upon turning RUNX1a-expression off and led to a loss of Sca1 expression (Lin- Kit+, LK). To further understand the molecular consequences of RUNX1a overexpression we sorted the LK cells and LSK cells before and 36h after RUNX1a-expression was turned off. Next, we performed microarray analysis to assess differential gene expression in these different subpopulations. Gene set enrichment analysis (GSEA) identified upregulation of genes highly expressed in hematopoietic stem cells (HSC) and leukemic stem cells (LSCs) in RUNX1a-expressing LSK cells compared to those LSK cells in which RUNX1a expression was turned off. Conversely, a gene signature associated with stemness and self-renewal was lost in LK-cells when RUNX1a expression was turned off. Among the eleven leading edge genes, we found genes implicated in leukemogenesis, stem cell regulation, or both such as Erg, Meis1 and Bcl11a. To further understand the role of RUNX1a in vivo we transplanted C57Bl6 mice (n=29) with HSPCs expressing RUNX1a in a competitive reconstitution setting. Consistent with the immortalization of HSPCs in vitro, RUNX1a-overexpressing HSPCs expanded in the bone marrow of transplanted mice. We observed significantly higher frequencies of LK (2.9-fold) and LSK cells (5-fold) in the RUNX1a-expressing bone marrow cells compared to transplanted control mice. High frequencies of RUNX1a-expressing cells in the bone marrow were associated with lower frequencies of RUNX1a-expressing cells in the peripheral blood indicating a differentiation block. In addition, we found that 85% of the RUNX1a-expressing cells were committed to the myeloid lineage (CD11b+/Ly6G+) at the expense of the lymphoid lineage (B220 and CD3e). Moreover, RUNX1a expression led to an increased percentage (65%) of immature erythroblasts (Ter119-) in the bone marrow compared to control cells (55%). In summary, we have demonstrated that RUNX1a overexpression immortalized HSPCs by upregulation of genes involved in leukemogenesis, stemness and self-renewal. In vivo such HSPCs showed a competitive advantage that was associated with a block of differentiation. Our study, particularly the gene expression analysis, provides novel insights into genetic drivers contributing to the development of myeloid malignancies in patients with RUNX1 mutations. Disclosures No relevant conflicts of interest to declare.

The Wnt signaling pathway plays a critical role in cell proliferation and differentiation, thus it is often associated with diseases such as cancers. Unfortunately, although attractive, developing anti-cancer strategy targeting Wnt signaling has been challenging given that the most attractive targets are involved in protein-protein interactions (PPIs). Here, we develop a stapled peptide inhibitor that targets the interaction between β-catenin and T cell factor/lymphoid enhancer-binding factor transcription factors, which are crucially involved in Wnt signaling. Our integrative approach combines peptide stapling to optimize proteolytic stability, with lessons learned from cell-penetrating peptide (CPP) design to maximize cellular uptake resulting in NLS-StAx-h, a selective, cell permeable, stapled peptide inhibitor of oncogenic Wnt signaling that efficiently inhibits β-catenin-transcription factor interactions. We expect that this type of integrative strategy that endows stapled peptides with CPP features will be generally useful for developing inhibitors of intracellular PPIs. Dietrich et al. investigate the cellular uptake of cell-penetrating peptides and stapled peptides. Based on their findings, a poorly permeable stapled peptide is converted into an excellent cell penetrator capable of efficiently inhibiting Wnt signaling. This agent selectively inhibits the growth and migration of Wnt-dependent cancer cells.

Summary Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1(−/−) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression., Highlights • HOXB4 induces hemogenic endothelium formation from differentiating ESCs • Hematopoiesis is not promoted at the cost of endothelial cell development • HOXB4 induces a transcriptional program closely resembling dorsal aorta HE cells, In this article, Klump and colleagues demonstrate that the human homeotic selector protein HOXB4 promotes ESC-derived hematopoiesis by inducing hemogenic endothelium formation, in vitro. It propels hematopoietic specification by upregulating the transcription of genes essential for hematopoietic development, such as those encoding members of the so-called heptad transcription factors.

Comprehensive analysis of the cancer genome has become a standard approach to identifying new disease loci, and ultimately will guide therapeutic decisions. A key technology in this effort, single nucleotide polymorphism arrays, has been applied in hematologic malignancies to detect deletions, amplifications, and loss of heterozygosity (LOH) at high resolution. An inherent challenge of such studies lies in correctly distinguishing somatically acquired, cancer-specific lesions from patient-specific inherited copy number variations or segments of homozygosity. Failure to include appropriate normal DNA reference samples for each patient in retrospective or prospective studies makes it difficult to identify small somatic deletions not evident by standard cytogenetic analysis. In addition, the lack of proper controls can also lead to vastly overestimated frequencies of LOH without accompanying loss of DNA copies, so-called copy-neutral LOH. Here we use examples from patients with myeloid malignancies to demonstrate the superiority of matched tumor and normal DNA samples (paired studies) over multiple unpaired samples with respect to reducing false discovery rates in high-resolution single nucleotide polymorphism array analysis. Comparisons between matched tumor and normal samples will continue to be critical as the field moves from high resolution array analysis to deep sequencing to detect abnormalities in the cancer genome.

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase II alpha-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

Progress in the management of patients with myelodysplastic syndromes (MDS) has been hampered by the inability to detect cytogenetic abnormalities in 40-60% of cases. We prospectively analyzed matched pairs of bone marrow and buccal cell (normal) DNA samples from 51 MDS patients by single nucleotide polymorphism (SNP) arrays, and identified somatically acquired clonal genomic abnormalities in 21 patients (41%). Among the 33 patients with normal bone marrow cell karyotypes, 5 (15%) had clonal, somatically acquired aberrations by SNP array analysis, including 4 with segmental uniparental disomies (UPD) and 1 with three separate microdeletions. Each abnormality was detected more readily in CD34+ cells than in unselected bone marrow cells. Paired analysis of bone marrow and buccal cell DNA from each patient was necessary to distinguish true clonal genomic abnormalities from inherited copy number variations and regions with apparent loss of heterozygosity. UPDs affecting chromosome 7q were identified in two patients who had a rapidly deteriorating clinical course despite a low-risk International Prognostic Scoring System score. Further studies of larger numbers of patients will be needed to determine whether 7q UPD detected by SNP array analysis will identify higher risk MDS patients at diagnosis, analogous to those with 7q cytogenetic abnormalities.

The growth of binary-alloy clusters on a weakly interacting substrate through codeposition of two atomic species is studied by kinetic Monte Carlo simulation. Our model describes salient features of CoPt 3 nanoclusters, as obtained recently by the molecular-beam epitaxy technique. The clusters display perpendicular magnetic anisotropy (PMA) in a temperature window of growth favorable for applications. This temperature window is found to arise from the interplay of Pt surface segregation and aspect ratio for cluster shapes. Conclusions are drawn on how to optimize growth parameters with respect to PMA.

We investigate ECG recordings of healthy subjects (H subjects) and those with heart failure (HF subjects) based on a new measure derived from the shape of the ECG signal during each heart beat. From this measure we construct sequences, called “morphograms”, which are complementary to sequences of successive interbeat time intervals, commonly called “tachograms”. The morphograms show long-range anticorrelations between the increments of their successive elements, analogous to the behavior found previously in the tachograms. By studying the average strength of the fluctuations of the increments on certain scales, we find that it is possible to completely separate the two groups of H and HF subjects. The separation is comparable, and in certain cases even more pronounced than that found earlier based on the tachograms.

Overexpression of HOXA9 is linked to the molecular pathogenesis of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS), conferring a poor prognosis. HOXA9 expression levels were analysed in the diagnostic bone marrow (BM) samples of 13 MDS patients. HOXA9 was expressed by CD34(+) BM cells at median levels 3.1-fold higher than in CD34(-) cells from the same patient and at median levels 4.3-fold higher than in CD34(+) cells from healthy donors. These results indicate that CD34(+) cell selection is required to accurately assess the expression levels of HOXA9 and related genes in the multipotential malignant progenitor cells of MDS patients.

Activation of the tumor suppressor p53 after genotoxic insults may result in two different responses: growth arrest or apoptosis. In this study, we analysed how mitogenic stimulation of primary mouse lymphocytes influences p53 signaling upon gamma-irradiation. We found that G(0) lymphocytes rapidly went into p53-dependent apoptosis, whereas stimulated lymphocytes went into a p53-dependent, p21-mediated growth arrest. The switch in p53 response upon stimulation did neither result from a switch in transcriptional activation of major p53 target genes, nor from the high level of p21 expressed in stimulated, irradiated cells. Growth stimulation, however, led to the upregulation of the antiapoptotic factors Bcl-x(L) and Bfl-1. In resting cells, p53 induced apoptosis after gamma-irradiation was accompanied by a breakdown of the mitochondrial membrane potential (psi(m)) that was counteracted by growth stimulation. We propose that growth stimulation intercepted p53 proapoptotic signaling at the level of mitochondrial integrity, most likely by upregulating the antiapoptotic factors Bcl-x(L) and Bfl-1. Upregulation of Bcl-x(L) and of Bfl-1 upon growth stimulation was mediated by the PKC-dependent activation of NF-kappaB. Consequently, blocking PKC activity restored apoptosis in stimulated, irradiated splenocytes. The inherent coupling of growth stimulation with antiapoptotic signaling in primary lymphocytes might provide hints as to how precancerous lymphocytes bypass the need for mutational inactivation of p53. Thus, our findings might explain the relatively low frequency of p53 mutations in lymphomas in comparison to other tumor entities.

We present kinetic Monte Carlo simulations of growing nanoclusters on a weakly interacting substrate within an fcc binary alloy model. This model is designed to describe essential structural and magnetic properties of CoPt 3 -type clusters grown by molecular beam epitaxy. Due to a growth-induced structural anisotropy located near the cluster surface, these clusters exhibit a perpendicular magnetic anisotropy in a certain temperature range of growth, in agreement with experiments. By applying an external magnetic field during the simulated growth, a clear numerical evidence for an enhanced perpendicular magnetic anisotropy in these systems is found under certain growth conditions.

Path-Integral-Monte-Carlo simulation has been used to calculate the properties of a two-dimensional (2D) interacting Bose system. The bosons interact with hard-core potentials and are confined to a harmonic trap. Results for the density profiles, the condensate fraction, and the superfluid density are presented. By comparing with the ideal gas we easily observe the effects of finite size and the depletion of the condensate because of interactions. The system is known to have no phase transition to a Bose-Einstein condensation in 2D, but the finite system shows that a significant fraction of the particles are in the lowest state at low temperatures.

A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20–30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these ‘sub-haploinsufficient’ cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies. DOI: http://dx.doi.org/10.7554/eLife.00825.001, eLife digest Blood cells are produced within bone marrow by specialized stem cells and progenitor cells. Abnormalities in this process lead to a group of diseases known as myeloid malignancies, which include acute myeloid leukaemia—in which the bone marrow produces abnormal white blood cells—and myelodysplastic syndromes, which are caused by too few mature blood cells being produced. Many individuals affected by these disorders possess a shortened form of chromosome 20 that lacks a number of genes. This deletion is only ever seen in one of their two copies of the chromosome—suggesting that at least some of these genes are essential for survival—but the identity of the gene(s) that are associated with the increased risk of myeloid malignancies is unknown. Now, Heinrichs et al. have uncovered a key tumor suppressor among those genes frequently lost on chromosome 20. The gene, which is called MYBL2, encodes a transcription factor that helps to control the cell division cycle. Myeloid malignancy patients lacking one copy of this gene showed levels of MYBL2 expression that were less than 50% of those in healthy individuals. This suggests that additional mechanisms must be acting to reduce expression of their remaining copy of the gene. Surprisingly, MYBL2 levels were also reduced in myeloid malignancy patients who possessed two intact copies of chromosome 20, indicating that loss of a single copy represents only one mechanism to reduce MYBL2 expression, i.e., the ‘tip-of-the-iceberg’. Hence, this finding reveals a more general role for MYBL2 as it indicates that more patients are likely to be affected by altered expression of this gene. To confirm their findings from studies in patients, Heinrichs et al. used gene silencing techniques to reduce the expression of MYBL2 in mice and showed that this induced symptoms of myeloid malignancies in the animals. Moreover, injection of modified cells from these animals into healthy mice also induced symptoms in the recipients. The modified cells are able to expand more robustly than normal cells, and this dominance induced by downregulation of the tumor suppressor increases the risk of malignancy. In addition to revealing a new tumor suppressor gene and its contribution to myeloid malignancies, the study by Heinrichs et al. highlights the importance of gene dosage in mediating the effects of tumor suppressors. DOI: http://dx.doi.org/10.7554/eLife.00825.002

Stochastic growth of binary alloys on a weakly interacting substrate is studied by kinetic Monte Carlo simulation. The underlying lattice model relates to fcc alloys, and the kinetics are based on deposition, atomic migration with bond-breaking processes, and exchange processes mediated by nearest neighbor hopping steps. We investigate the interrelation between surface processes and the emerging nonequilibrium structure at and below the growing surface under conditions where atoms in the bulk can be regarded as immobile. The parameters of the model are adapted to ${\mathrm{CoPt}}_{3}$ alloys. Growing nanoclusters exhibit an anisotropic short range order, primarily caused by Pt segregation at the surface. The overall structural anisotropy depends on both Pt surface segregation and cluster shape, and can explain the perpendicular magnetic anisotropy (PMA) recently measured in ${\mathrm{CoPt}}_{3}$ nanoclusters on a van der Waals substrate. The onset of $L{1}_{2}$ ordering in the cluster is induced by surface processes. The same kinetic model is applied also to continuous thin films, which in addition can exhibit a small bulk contribution to PMA.

The statistical properties of heart beat intervals of 130 long-term surface electrocardiogram recordings during atrial fibrillation (AF) are investigated. We find that the distribution of interbeat intervals exhibits a characteristic exponential tail, which is absent during sinus rhythm, as tested in a corresponding control study with 72 healthy persons. The rate of the exponential decay lies in the range 3-12 Hz and shows diurnal variations. It equals, up to statistical uncertainties, the level of the previously uncovered white noise part in the power spectrum, which is also characteristic for AF. The overall statistical features can be described by decomposing the intervals into two statistically independent times, where the first one is associated with a correlated process with 1/f noise characteristics, while the second one belongs to an uncorrelated process and is responsible for the exponential tail. It is suggested to use the rate of the exponential decay as a further parameter for a better classification of AF and for the medical diagnosis. The relevance of the findings with respect to a general understanding of AF is pointed out.

Using the method of local Continuous Detrended Fluctuation Analysis CDFA) we analyze the correlations of ventricular interbeat intervals of patients with Atrial Fibrillation (AF). CDFA yields a local Hoelder exponent h for a neighborhood around each point in the time series by determining the scaling of fluctuations with window size after detrending. We compare the histograms of Hoelder exponents for original data with those of randomly shuffled data and find some correlations not only in long-range windows but also at short time scales where interbeat intervals during AF have been believed to be random in nature. Furthermore, we find unique temporal correlation structures to occur only in the heart rate of patients who were in the survivor group when a follow up was conducted at least one year after data acquisition. We conclude that ventricular interbeat intervals during AF contain richer information than previously considered and the study of the local correlations may be useful in predicting mortality of the patients.

We present an extension of the density-functional theory (DFT) formalism for lattice gases to systems with internal degrees of freedom. In order to test approximations commonly used in DFT approaches, we investigate the statics and dynamics of occupation (density) profiles in the one-dimensional Potts model. In particular, by taking the exact functional for this model we can directly evaluate the quality of the local equilibrium approximation used in time-dependent density-functional theory (TDFT). Excellent agreement is found in comparison with Monte Carlo simulations. Finally, principal limitations of TDFT are demonstrated., Comment: 8 pages, 3 figures

We develop a theory for the inclusion of adatom interactions in second layer nucleation occurring in epitaxial growth. The interactions considered are due to ring barriers between pairs of adatoms and binding energies of unstable clusters. The theory is based on a master equation, which describes the time development of microscopic states that are specified by cluster configurations on top of an island. The transition rates are derived by scaling arguments and tested against kinetic Monte-Carlo simulations. As an application we reanalyze experiments to determine the step edge barrier for Ag/Pt(111)., Comment: 4 pages, 4 figures

A Comment on the Letter by K. R. Roos and M. C. Tringides, Phys. Rev. Lett. 85, 1480 (2000). The authors of the Letter offer a Reply.

We develop a theory for nucleation on top of islands in epitaxial growth based on the derivation of lifetimes and rates governing individual microscopic processes. These processes are strongly affected by the additional step edge barrier E_S for descending atoms. If the dissociation times of unstable clusters can be neglected, the theory predicts that for small critical nuclei of size i=3 by contrast, the nucleation process can be described in a mean-field type manner, which for large E_S corresponds to the approach developed by Tersoff et al. [Phys. Rev. Lett. 72, 266 (1994)]. In both the fluctuation-dominated and the mean-field case, various scaling regimes are identified, where the typical island size at the onset of nucleation shows a power law in dependence on the adatom diffusion rates, the incoming atom flux, and the step edge crossing probability exp(-E_S/k_B T). For the case where the dissociation rates of metastable clusters enter the problem as additional parameters, we set up a semi-analytical approach based on novel rate equations, which can easily be solved numerically. The results obtained from the theory are in good agreement with Monte Carlo data. Implications for various applications are pointed out., 24 pages, 10 figures

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (delta-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of delta-area and number of AgNORs. Various AgNOR parameters (CV of number, CV of delta-area, CV of area, mean delta-area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.

Abstract 1865 Cytogenetic changes, mainly deletions, can be found in about 30–50% of patients with Myelodysplastic Syndromes (MDS). To identify a tumor suppressor candidate within a commonly deleted region on chromosome 20q, we performed gene expression analysis on CD34+ bone marrow cells obtained from 8 patients with a 20q aberration and 18 with a normal karyotype. However, we were unable to identify genes that were significantly differentially expressed in aberrant 20q karyotype as compared to normal karyotype MDS patients. In contrast, a comparison of CD34+ cells from all MDS cases analyzed (n=26) with CD34+ cells obtained from normal bone marrow (n=4) revealed 108 genes that were differentially expressed. Interestingly, one of the top-scoring genes was MYBL2, which is located on chromosome 20q. MYBL2 levels were downregulated more than two-fold in 18 out of 26 cases. RNAi-mediated knockdown of MYBL2 in CD34+ normal bone marrow cells revealed a signature of genes functionally associated with the G2/M cell cycle phase confirming the well-documented role of MYBL2 as key transcription factor governing the onset of cell division. We hypothesize that in a subset of MDS cases the control of cell division may be impaired by low levels of MYBL2 such that altered cell fates established during cell division in early hematopoietic stem and progenitor cells will lead to clonal expansion with imbalanced or impaired differentiation. Indeed, gene set enrichment analysis revealed a strong enrichment of MYBL2 signature genes in MDS CD34+ cells. In support of a potential role as tumor suppressor, resequencing of MYBL2 (144 patients) identified 2 somatic mutations, pinpointing an additional mechanism to reduce expression of normal levels of wild-type MYBL2. Disclosures: No relevant conflicts of interest to declare.

In path integral Monte Carlo (PIMC) treatments of harmonically trapped bosons, one cannot use the usual long-range constant limit of the one-body reduced density matrix ρ 1 to determine the condensate number n 0 because ρ 1 always approaches zero in a trap. W. Krauth found that the longest permutation cycle arising in the simulation gives a consistent value of n 0 . Our analytical studies of the ideal gas suggest other ways of using permutation cycles to determine n 0 . We test these approaches on simulations involving finite-size ideal and interacting gases and find that the methods are consistent.

Abstract 277 NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures: No relevant conflicts of interest to declare.

Myelodysplastic syndromes (MDS) comprise a large group of hematological diseases sharing clinical features, but the diverse mechanisms underlying their molecular pathogenesis remain largely undefined. In this study, we used high-density 250K SNP arrays to analyze matched constitutional (buccal) and whole bone marrow DNAs from 36 patients with MDS. In 14 cases, we also selected CD34+ stem and progenitor cells for comparison. 23 of the cases were cytogenetically normal. Whereas regions of genomic amplification were rare, we found distinct regions of heterozygous deletion in 9 patients, most frequently targeting chromosomes 5q, 7q, 17p and 20q. In one patient with a large heterozygous 5q deletion, a smaller region in 5q34 was homozygously lost. Based on genotyping we could identify regions of uniparental disomy (UPD) in 4 patients, three of whom had normal bone marrow karyotypes. Two of the regions of UPD targeted chromosome 7q, suggesting that the frequency of 7q deletion is underestimated by conventional cytogenetics, and that a mutation has occurred in these cases within the reduplicated region involved in UPD. Comparison with constitutional DNA was critical for accurate SNP analysis in MDS, allowing us to focus on acquired clonal abnormalities and control for changes due to inherited copy number variation or unusual regions of germline homozygosity. For example, one case had several large blocks of germline homozygosity distributed over various chromosomes, and by comparison with the buccal DNA we were able to discern that these regions did not result from clonal somatic abnormalities associated with MDS. The analysis of CD34+ cellular DNA in 14 cases revealed that all aberrations identified in the stem and progenitor fraction were also identified in whole bone marrow DNA. This finding emphasizes the dominance and stem cell involvement of the abnormal clone in MDS, which makes purification of CD34 cells unnecessary for SNP array analysis in this disease. Most, but not all, clonal deletions evident by cytogenetic analysis were detected as copy number alterations by SNP array, possibly indicating that such abnormalities occurred in subpopulations of the cytogenetically analyzed MDS cells. Thus, SNP array and cytogenetic analysis are complementary methods for the identification of abnormal clones in MDS patients, each detecting overlapping but distinct subsets of patients. Our data indicate that SNP array analysis should be performed in all patients with MDS and normal bone marrow cytogenetics, in order to help discriminate patients with UPD indicating cell autonomous clonal stem cell malignancy from those who might have extrinsically mediated bone marrow failure syndromes.

SNP microarrays are a powerful way of uncovering mutations relevant to cancer., Recent studies using single-nucleotide polymorphism arrays have pinpointed novel oncogenes and tumor suppressors involved in specific types of human cancers.

Kinetic Monte Carlo simulations are performed to study the influence of external magnetic fields on the growth of magnetic fcc binary alloy nanoclusters with perpendicular magnetic anisotropy. The underlying kinetic model is designed to describe essential structural and magnetic properties of CoPt_3-type clusters grown on a weakly interacting substrate through molecular beam epitaxy. The results suggest that perpendicular magnetic anisotropy can be enhanced when the field is applied during growth. For equilibrium bulk systems a significant shift of the onset temperature for L1_2 ordering is found, in agreement with predictions from Landau theory. Stronger field induced effects can be expected for magnetic fcc-alloys undergoing L1_0 ordering., 10 pages, 3 figures

MEIS proteins constitute an important subgroup of three amino acid loop extension (TALE) class transcription factors, which are characterized by an atypical homeodomain. MEIS1 has been shown to act as an important cofactor in HOXA9-mediated leukemogenesis in bone marrow transduction experiments. In human leukemia, MEIS1 was identified as a gene consistently upregulated in leukemias with MLL translocations. However, MEIS1 overexpression is also frequently found in AMLs with normal or complex karyotypes. To investigate the role of MEIS1 in AML pathogenesis, we measured the expression of MEIS1 and MEIS2 in 26 different established human AML cell lines, and documented high levels of expression of one or both genes in 20 of these lines. Despite the prevalence of MEIS1 expression in AML blasts, the downstream pathways and their contribution to the proliferation or self-renewal of the malignant clone are not well understood. To identify gene expression programs controlled by MEIS1, we targeted MEIS1 expression by RNAi in two AML cell lines that express HOXA9 and MEIS1, but only low levels of MEIS2. In comparison to a control shRNA, cells expressing two different MEIS1-specific shRNAs were markedly deficient in thymidine incorporation indicating very low levels of cellular proliferation. The RNAi-mediated knock-down of MEIS1 expression resulted in down-regulation of the tyrosine kinase receptor FLT3 by quantitative RT-PCR. Thus our data in human AML is consistent with results of Wang et al. (Blood, 106(1):254–64) implicating MEIS1 in the regulation of FLT3 tyrosine kinase expression, based on overexpression studies in mouse bone marrow progenitors. Our results in human AML suggest that MEIS1 is required for high levels of FLT3 expression, which is significant because FLT3 is mutated and activated in these two myeloid leukemia cell lines, suggesting the hypothesis that FLT3 is an important component of the pathway through which MEIS1 promotes proliferation. Programmed re-expression of activated FLT3 in AML lines with MEIS knock-down is underway to test whether FLT3 is the essential component acting downstream of MEIS1 expression in AML pathogenesis.

The discovery that Slug has a vital anti-apoptotic role in the normal response of hematopoietic progenitors to genotoxic stress compelled us to investigate the underlying cellular and molecular mechanism(s) of this effect. Here we show that, although the development of myeloid progenitors in Slug−/− mice is not impaired under steady-state conditions, their ability to repopulate the compartment after γ-irradiation is reduced. We demonstrate that the radiation-induced death of Slug−/− mice exclusively reflects bone marrow failure and show that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. We also establish that Slug confers radioprotection by inhibiting the mitochondria-dependent apoptotic pathway activated by γ-irradiation in hematopoietic progenitors. We show that Slug acts as a transcriptional repressor by directly antagonizing p53-mediated upregulation of the BH3-only gene Puma, which was recently shown to encode a critical mediator of p53-induced apoptosis. Finally, we provide evidence for a novel feedback loop in the hematopoietic progenitor cells after DNA damage: Slug is itself induced by p53. Thus, the survival of hematopoietic progenitor cells after genotoxic stress relies on induction of Slug by p53 and Slug-mediated repression of Puma.

Studies in mice and humans suggests that HOX genes integrate the myelodysplastic and leukemogenic activities of different oncogenes. HOXA9 has been identified as a major downstream target in patients with MLL translocations and amplifications. Since upregulation of HOXA9 and other specific HOX genes is more frequent than MLL deregulation, additional upstream pathways have to be postulated. Given the fact that HOXA9 is targeted by several yet to be identified mechanisms, we asked whether other members of the HOX9 paralog group are also highly expressed in myeloid malignancies. Microarray gene expression analysis of 449 AML patients revealed that besides HOXA9 (61% of all cases), HOXB9 is upregulated in 15% of the cases, whereas the overexpression of HOXC9 and HOXD9 expression is a very rare event (>1% of all cases). High levels of HOXB9 expression did not correlate with low levels of HOXA9 expression ruling out the possibility that HOXB9 compensates for a lack of HOXA9 activity in the leukemogenesis of a HOXA9 negative AML cases. Moreover, within the cytogenetically defined subgroups inv(16), t(15;17), t(8;21) that have no HOXA9 expression at all, HOXB9 was also low or absent. The analysis of 27 AML cell lines revealed a gene expression pattern of HOXA9 and HOXB9 that reflected the frequency of overexpression found in patient samples (14/27 showed HOXA9 expression; 6/27 showed HOXB9 expression). Interestingly, the absolute expression level of HOXB9 mRNA in cell lines is about 10fold higher than HOXA9 as measured by real-time RT-PCR. Western-blotting with an antibody recognizing both HOXA9 and HOXB9 confirmed this result on the protein level. However, the analysis of the absolute levels in normal CD34+ bone marrow cells revealed an inverse ratio, with very low levels of HOXB9. Taken together these results indicate that HOXB9 expression is aberrant in the leukemic clone. To identify downstream pathways driven by HOXB9 overexpression we designed two highly efficient siRNAs targeting HOXB9 and knocked down its expression by retroviral transduction. In comparison to cells expressing a control siRNA, the resulting cell lines showed Our results indicate that aberrant expression of HOXB9 inhibits the differentiation of leukemic myeloid progenitor cells and maintains the cells of the leukemic clone in an undifferentiated and rapidly proliferative state.

Emerging evidence from studies in mice and humans suggests that HOX genes integrate the myelodysplastic and leukemogenic activities of different oncogenes. Translocations involving the MLL gene, a major upstream regulator of the HOXA cluster, have been identified in AML and define a specific cytogenetic subgroup of this disease. Recently, amplification of MLL was found in AML and MDS cases, revealing a second mechanism of deregulation of MLL and its downstream targets. However, additional leukemogenic upstream pathways have to be postulated, since upregulation of specific HOX genes, particularly HOXA9, is more frequent than MLL deregulation. Given the fact that HOXA9 is targeted by several yet to be identified mechanisms, we asked whether other members of the HOX9 paralog group are also highly expressed in myeloid malignancies. Our analysis of 449 AML patients by gene expression microarray revealed that besides HOXA9 (61% of all cases), HOXB9 is upregulated in 15% of the cases, whereas the overexpression of HOXC9 and HOXD9 expression is a very rare event (>1% of all cases). High levels of HOXB9 expression did not correlate with low levels of HOXA9 expression ruling out the possibility that HOXB9 compensates for a lack of HOXA9 activity in the leukemogenesis of a HOXA9 negative AML cases. Interestingly, within the cytogenetically defined subgroups (inv(16), t(15;17), t(8;21)) that have no HOXA9 expression at all, HOXB9 was also low or absent. To test if HOXB9 has the potential to contribute to the leukemogenic phenotype we analyzed a panel of human AML cell lines. The finding that 6 out of 24 cell lines express high levels of HOXB9 further indicates the significance of HOXB9 in myeloid malignancies. We chose K-562 and HEL as model cell lines since they express high levels of HOXB9, but lack HOXA9 expression. To reveal HOXB9 dependent pathways we designed two highly efficient siRNAs to target HOXB9 and knocked down its expression by retroviral transduction. In comparison to cells expressing a control siRNA, the resulting cell lines showed

An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.

In recent years, it has become increasingly clear that overexpression of specific major HOX genes is linked to transformation of the malignant hematopoietic stem cells underlying human acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). For example, several recent studies indicate that overexpression of HOXA9 is associated with a poor outcome in AML and MDS. As we initiated our own studies of HOXA9 expression in MDS, we were concerned about the validity of studying whole bone marrow (BM) cell RNA obtained at diagnosis, due to the diversity of cell types and low blast cell numbers in the BM that characterize MDS, and prior studies of G. Savageau and K. Humphries demonstrating that HOXA9 expression in normal BM cells is limited to the CD34+ fraction, which is enriched for hematopoietic stem and progenitor cells. Thus, we initiated this study to investigate whether valid HOXA9 expression levels can be determined from whole BM cell RNAs of MDS patients or whether CD34+ cell separation is required prior to analysis. BM aspirate samples were collected at diagnosis from 9 patients with MDS and subjected to Ficoll-Hypaque light density cell separation, followed by magnetic bead separation to remove CD3+/CD19+ mature lymphoid cells. The CD3−/CD19− fraction was further fractionated into CD34+ and CD34− populations. RNA was extracted by standard methods and two-step quantitative real-time RT-PCR was performed using SYBR Green to measure HOXA9 and control housekeeping gene expression levels. RNAs from peripheral blood CD34+ cells from 8 healthy donors served as controls. Relative HOXA9 expression levels were obtained by normalization to three housekeeping genes (GAPDH, YWHAZ, and RPL4) for each fraction. Our results conclusively show that HOXA9 levels of CD34+ cells from MDS patients are on average three-fold higher than those of CD34− cells from the same patient (p=0.004, Wilcoxon signed rank). Thus, as is the case for normal BM cells, HOXA9 expression in MDS is highest in the stem and progenitor cell fraction. Our results indicate that CD34+ cell selection must be carried out to accurately assess the expression of HOXA9 in the malignant clone of MDS patients. Unselected BM cell measurements include variable fractions of CD34− and CD34+ cells, which can have significant variability, even in samples obtained from the same patient, thus confounding attempts to accurately measure HOXA9 levels and determine the contribution of this oncogene to MDS. Moreover, we detected levels of HOXA9 expression in the CD34+ cells of MDS patients that were on average 4.3 times those of normal controls (p=0.004, Wilcoxon rank sum). Our studies indicate that in order to advance understanding of the molecular pathogenesis of MDS, the expression levels of specific HOX genes and their co-regulators will need to be carefully evaluated in purified CD34+ BM cells from patients with this disease.

SummaryIn response to DNA damage, the p53 tumor suppressor can elicit either apoptosis or cell-cycle arrest and repair, but how this critical decision is made in specific cell types remains largely undefined. We investigated the mechanism by which the transcriptional repressor Slug specifically rescues hematopoietic progenitor cells from lethal doses of γ radiation. We show that Slug is transcriptionally induced by p53 upon irradiation and then protects the damaged cell from apoptosis by directly repressing p53-mediated transcription of puma, a key BH3-only antagonist of the antiapoptotic Bcl-2 proteins. We established the physiologic significance of Slug-mediated repression of puma by demonstrating that mice deficient in both genes survive doses of total-body irradiation that lethally deplete hematopoietic progenitor populations in mice lacking only slug. Thus, Slug functions downstream of p53 in developing blood cells as a critical switch that prevents their apoptosis by antagonizing the trans-activation of puma by p53.

The possible application of the combination of horseradish peroxidase (HRP) and Lucifer Yellow CH (LY) for differential retrograde labelling of a relatively large invertebrate nervous system requiring the enzymatic marker to be processed after fixation and sectioning was evaluated. In control experiments an HRP fluorescence induced by glutaraldehyde (GA) fixation was detected and its spectral properties were investigated. This fluorescence, however, does not occur after previous treatment of the tissue with depolymerized paraformaldehyde (PFA) instead of GA. PFA as a fixing agent is also necessary to yield maximum preservation of LY labelling during subsequent HRP histochemistry. Both findings strongly recommend the use of PFA instead of GA as fixative for the combination of HRP and LY. It is shown that the HRP/LY differential labelling technique renders a valid method for the study of the structural interactions at the fibre level of identified neurones in a relatively large invertebrate nervous system, also after retrograde tracing.

Myelopoiesis is a developmental process through which stem cells give rise to mature monocytes and granulocytes by means of a tightly regulated cellular program. The molecular mechanisms controlling myelopoiesis are incompletely understood, as are the abnormalities in this process that give rise to myeloid cell diseases, such as leukemia, myelodysplasia, and myeloproliferative syndromes. These diseases are commonly associated with somatically acquired large chromosomal deletions, making the identification of the causative tumor suppressor genes difficult. To learn more about the genes required for normal myelopoiesis, which we postulated might be mutated during the molecular pathogenesis of myeloid malignancies, we performed a screen using a panel of insertional mutants provided by Nancy Hopkins (MIT) to identify zebrafish lines with deficiencies in myeloperoxidase (mpo), a gene specifically expressed in granulocytes. As part of this screen, we identified the line hi2648, in which homozygous mutants have a severe reduction in the numbers of mature myeloid cells expressing either mpo or l-plastin, and in which the small numbers of residual myeloid cells are abnormally large. We bred hi2648 mutant fish to a line carrying the pu.1:GFP transgene and found that the homozygous mutant embryos displayed decreased numbers but relatively large GFP-positive cells, which were also observed in cytospins of FACS-purified, GFP-expressing cells, consistent with our in situ analysis. Hi2648 harbors a retroviral insertion that disrupts the gene emi1 between exons one and two, resulting in decreased levels of emi1 RNA assayed by whole- mount in situ analysis. We confirmed that the hematopoietic phenotype in this line is due to disruption of emi1 by showing that we could phenocopy the mutant using emi1-specific morpholinos and also by rescuing the mutant phenotype using forced expression of emi1 RNA. The Emi1 gene product has been shown to inhibit anaphase-promoting complex during interphase of the cell cycle. We analyzed emi1-mutant embryos for cell cycle defects by FACS analysis of propidium-iodide stained cells at 21 hpf, and showed that impaired emi1 expression results in an accumulation of cells in G2 phase, presumably due to premature activation of APC during S- and G2-phase with an inability to accumulate adequate levels cyclin B. We also observed a range of increased of levels of cellular DNA content greater than the normal 4n complement, suggesting that loss of emi1 also promotes abortive endoreduplication. Mutational analysis indicated gene-specific disruption in of emi1 in a human leukemia cell line, suggesting that this gene represents one of the long-sought haploinsufficient tumor suppressors located on the long arm of human chromosome 6 (del 6q), whose loss promotes genomic instability during leukemogenesis and the progression of MDS to AML in man.