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Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro , resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro , which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis. IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies., (Copyright © 2020 American Society for Microbiology.)

The prognosis for many patients with acute myeloid leukemia (AML) is poor, mainly due to disease relapse driven by leukemia stem cells (LSCs). Recent studies have highlighted the unique metabolic properties of LSCs, which might represent opportunities for LSC-selective targeting. LSCs characteristically have low levels of reactive oxygen species (ROS), which apparently result from a combination of low mitochondrial activity and high activity of ROS-removing pathways such as autophagy. Due to this low activity, LSCs are highly dependent on mitochondrial regulatory mechanisms. These include the anti-apoptotic protein BCL-2, which also has crucial roles in regulating the mitochondrial membrane potential, and proteins involved in mitophagy. Here we review the different pathways that impact mitochondrial activity and redox-regulation, and highlight their relevance for the functionality of both HSCs and LSCs. Additionally, novel AML therapy strategies that are based on interference with those pathways, including the promising BCL-2 inhibitor Venetoclax, are summarized., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

PKAN, CoPAN, MePAN, and PDH-E2 deficiency share key phenotypic features but harbor defects in distinct metabolic processes. Selective damage to the globus pallidus occurs in these genetic neurodegenerative diseases, which arise from defects in CoA biosynthesis (PKAN, CoPAN), protein lipoylation (MePAN), and pyruvate dehydrogenase activity (PDH-E2 deficiency). Overlap of their clinical features suggests a common molecular etiology, the identification of which is required to understand their pathophysiology and design treatment strategies. We provide evidence that CoA-dependent activation of mitochondrial acyl carrier protein (mtACP) is a possible process linking these diseases through its effect on PDH activity. CoA is the source for the 4'-phosphopantetheine moiety required for the posttranslational 4'-phosphopantetheinylation needed to activate specific proteins. We show that impaired CoA homeostasis leads to decreased 4'-phosphopantetheinylation of mtACP. This results in a decrease of the active form of mtACP, and in turn a decrease in lipoylation with reduced activity of lipoylated proteins, including PDH. Defects in the steps of a linked CoA-mtACP-PDH pathway cause similar phenotypic abnormalities. By chemically and genetically re-activating PDH, these phenotypes can be rescued, suggesting possible treatment strategies for these diseases., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Ring sideroblasts (RS) emerge as result of aberrant erythroid differentiation leading to excessive mitochondrial iron accumulation, a characteristic feature for myelodysplastic syndromes (MDS) with mutations in the spliceosome gene SF3B1. However, RS can also be observed in patients diagnosed with acute myeloid leukemia (AML). The objective of this study was to characterize RS in patients with AML. Clinically, RS-AML is enriched for ELN adverse risk (55%). In line with this finding, 35% of all cases had complex cytogenetic aberrancies, and TP53 was most recurrently mutated in this cohort (37%), followed by DNMT3A (26%), RUNX1 (25%), TET2 (20%), and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole-exome sequencing and SNP array analysis on a subset of patients did not uncover a single genetic defect underlying the RS phenotype. Shared genetic defects between erythroblasts and total mononuclear cell fraction indicate common ancestry for the erythroid lineage and the myeloid blast cells in patients with RS-AML. RNA sequencing analysis on CD34+ AML cells revealed differential gene expression between RS-AML and non RS-AML cases, including genes involved in megakaryocyte and erythroid differentiation. Furthermore, several heme metabolism-related genes were found to be upregulated in RS- CD34+ AML cells, as was observed in SF3B1mut MDS. These results demonstrate that although the genetic background of RS-AML differs from that of RS-MDS, they have certain downstream effector pathways in common., (© 2019 by The American Society of Hematology.)

Reduced expression of the transcription factor PU.1 is frequently associated with development of acute myeloid leukemia (AML), whereas elevated levels of CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) enhance maintenance of both normal and leukemic hematopoietic stem and progenitor cells (HSPCs). Recent findings indicate that PU.1 and CITED2 act in the same gene regulatory network. We therefore examined a potential synergistic effect of simultaneous PU.1 downregulation and CITED2 upregulation on stem cell biology and AML pathogenesis. We found that simultaneous PU.1/CITED2 deregulation in human CD34 + cord blood (CB) cells, as well as CITED2 upregulation in preleukemic murine PU.1-knockdown (PU.1 KD/KD ) bone marrow cells, significantly increased the maintenance of HSPCs compared with the respective deregulation of either factor alone. Increased replating capacity of PU.1 KD/KD /CITED2 cells in in vitro assays eventually resulted in outgrowth of transformed cells, while upregulation of CITED2 in PU.1 KD/KD cells enhanced their engraftment in in vivo transplantation studies without affecting leukemic transformation. Transcriptional analysis of CD34 + CB cells with combined PU.1/CITED2 alterations revealed a set of differentially expressed genes that highly correlated with gene signatures found in various AML subtypes. These findings illustrate that combined PU.1/CITED2 deregulation induces a transcriptional program that promotes HSPC maintenance, which might be a prerequisite for malignant transformation., (Copyright © 2019. Published by Elsevier Inc.)

In this study, we demonstrate that, among all five CBX Polycomb proteins, only CBX7 possesses the ability to control self-renewal of human hematopoietic stem and progenitor cells (HSPCs). Xenotransplantation of CBX7-overexpressing HSPCs resulted in increased multi-lineage long-term engraftment and myelopoiesis. Gene expression and chromatin analyses revealed perturbations in genes involved in differentiation, DNA and chromatin maintenance, and cell cycle control. CBX7 is upregulated in acute myeloid leukemia (AML), and its genetic or pharmacological repression in AML cells inhibited proliferation and induced differentiation. Mass spectrometry analysis revealed several non-histone protein interactions between CBX7 and the H3K9 methyltransferases SETDB1, EHMT1, and EHMT2. These CBX7-binding proteins possess a trimethylated lysine peptide motif highly similar to the canonical CBX7 target H3K27me3. Depletion of SETDB1 in AML cells phenocopied repression of CBX7. We identify CBX7 as an important regulator of self-renewal and uncover non-canonical crosstalk between distinct pathways, revealing therapeutic opportunities for leukemia., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; P = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future., (© 2018 by The American Society of Hematology.)

CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) is a regulator of the acetyltransferase CBP/p300 and elevated CITED2 levels are shown in a number of acute myeloid leukemia (AML). To study the in vivo role of CITED2 in AML maintenance, AML cells were transduced with a lentiviral construct for RNAi-mediated knockdown of CITED2. Mice transplanted with CITED2-knockdown AML cells (n=4) had a significantly longer survival compared to mice transplanted with control AML cells (P<0.02). In vitro, the reduction of CITED2 resulted in increased p53-mediated apoptosis and CDKN1A expression, whereas BCL2 levels were reduced. The activation of p53 upon CITED2 knockdown is not a direct consequence of increased CBP/p300-activity towards p53, since no increased formation of CBP/p300/p53 complexes was demonstrated and inhibition of CBP/p300-activity could not rescue the phenotype of CITED2-deficient cells. Instead, loss of CITED2 had an inhibitory effect on the AKT-signaling pathway, which was indicated by decreased levels of phosphorylated AKT and altered expression of the AKT-pathway regulators PHLDA3 and SOX4. Notably, simultaneous upregulation of BCL2 or downregulation of the p53-target gene PHLDA3 rescued the apoptotic phenotype in CITED2-knockdown cells. Furthermore, knockdown of CITED2 led to a decreased interaction of p53 with its inhibitor MDM2, which results in increased amounts of total p53 protein. In summary, our data indicate that CITED2 functions in pathways regulating p53 activity and therefore represents an interesting target for AML therapy, since de novo AML cases are characterized by an inactivation of the p53 pathway or deregulation of apoptosis-related genes.

Background: Serologic determination of the Vel- phenotype is challenging due to variable Vel expression levels. In this study we investigated the genetic basis for weak Vel expression levels and developed a high-throughput genotyping assay to detect Vel- donors., Study Design and Methods: In 548 random Caucasian and 107 Vel+(w) donors genetic variation in the SMIM1 gene was studied and correlated to Vel expression levels. A total of 3366 Caucasian, 621 black, and 333 Chinese donors were screened with a high-throughput genotyping assay targeting the SMIM1*64&#95;80del allele., Results: The Vel+(w) phenotype is in most cases caused by the presence of one SMIM1 allele carrying the major allele of the rs1175550 SNP in combination with a SMIM1*64&#95;80del allele or in few cases caused by the presence of the SMIM1*152T>A or SMIM1*152T>G allele. In approximately 6% of Vel+(w) donors genetic factors in SMIM1 could not explain the weak expression. We excluded the possibility that lack of expression of another blood group system was correlated with weak Vel expression levels. Furthermore, using a high-throughput Vel genotyping assay we detected two Caucasian Vel- donors., Conclusion: Weak Vel expression levels are caused by multiple genetic factors in SMIM1 and probably also by other genetic or environmental factors. Due to the variation in Vel expression levels, serologic determination of the Vel- phenotype is difficult and a genotyping assay targeting the c.64&#95;80del deletion in SMIM1 should be used to screen donors for the Vel- phenotype., (© 2015 AABB.)

Development and maintenance of leukemia can be partially attributed to alterations in (anti)-apoptotic gene expression. Genome-wide transcriptome analyses revealed that 89 apoptosis-associated genes were differentially expressed between patient acute myeloid leukemia (AML) CD34(+) cells and normal bone marrow (NBM) CD34(+) cells. Among these, transforming growth factor-β activated kinase 1 (TAK1) was strongly upregulated in AML CD34(+) cells. Genetic downmodulation or pharmacologic inhibition of TAK1 activity strongly impaired primary AML cell survival and cobblestone formation in stromal cocultures. TAK1 inhibition was mainly due to blockade of the nuclear factor κB (NF-κB) pathway, as TAK1 inhibition resulted in reduced levels of P-IκBα and p65 activity. Overexpression of a constitutive active variant of NF-κB partially rescued TAK1-depleted cells from apoptosis. Importantly, NBM CD34(+) cells were less sensitive to TAK1 inhibition compared with AML CD34(+) cells. Knockdown of TAK1 also severely impaired leukemia development in vivo and prolonged overall survival in a humanized xenograft mouse model. In conclusion, our results indicate that TAK1 is frequently overexpressed in AML CD34(+) cells, and that TAK1 inhibition efficiently targets leukemic stem/progenitor cells in an NF-κB-dependent manner., (© 2014 by The American Society of Hematology.)

With the emergence of the concept of the leukemic stem cell (LSC), assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID or NSG xenotransplantation model is currently still the favored assay of choice in most cases, this system has some limitations as well such as its cost-effectiveness, duration, and lack of engraftability of cells from some acute myeloid leukemia (AML) patients. Here, we describe in vitro assays in which long-term expansion and self-renewal of LSCs isolated from AML patients can be evaluated. We have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in primary AML cells, and these approaches are described in detail here. Also, we describe bone marrow stromal coculture systems in which cobblestone area-forming cell activity, self-renewal, long-term expansion, and in vitro myeloid or lymphoid transformation can be evaluated in human CD34(+) cells of fetal or adult origin that are engineered to express oncogenes. Together, these tools should allow a further molecular elucidation of derailed signal transduction in LSCs.

· Diets rich in broccoli (Brassica oleracea var italica) have been associated with maintenance of cardiovascular health and reduction in risk of cancer. These health benefits have been attributed to glucoraphanin that specifically accumulates in broccoli. The development of broccoli with enhanced concentrations of glucoraphanin may deliver greater health benefits. · Three high-glucoraphanin F1 broccoli hybrids were developed in independent programmes through genome introgression from the wild species Brassica villosa. Glucoraphanin and other metabolites were quantified in experimental field trials. Global SNP analyses quantified the differential extent of B. villosa introgression · The high-glucoraphanin broccoli hybrids contained 2.5-3 times the glucoraphanin content of standard hybrids due to enhanced sulphate assimilation and modifications in sulphur partitioning between sulphur-containing metabolites. All of the high-glucoraphanin hybrids possessed an introgressed B. villosa segment which contained a B. villosa Myb28 allele. Myb28 expression was increased in all of the high-glucoraphanin hybrids. Two high-glucoraphanin hybrids have been commercialised as Beneforté broccoli. · The study illustrates the translation of research on glucosinolate genetics from Arabidopsis to broccoli, the use of wild Brassica species to develop cultivars with potential consumer benefits, and the development of cultivars with contrasting concentrations of glucoraphanin for use in blinded human intervention studies., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.

The level of transcription factor activity critically regulates cell fate decisions such as hematopoietic stem cell self-renewal and differentiation. The balance between hematopoietic stem cell self-renewal and differentiation needs to be tightly controlled, as a shift toward differentiation might exhaust the stem cell pool, while a shift toward self-renewal might mark the onset of leukemic transformation. A number of transcription factors have been proposed to be critically involved in governing stem cell fate and lineage commitment, such as Hox transcription factors, c-Myc, Notch1, β-catenin, C/ebpα, Pu.1 and STAT5. It is therefore no surprise that dysregulation of these transcription factors can also contribute to the development of leukemias. This review will discuss the role of STAT5 in both normal and leukemic hematopoietic stem cells as well as mechanisms by which STAT5 might contribute to the development of human leukemias.

The regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches, we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast, conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16(Ink4a) and p19(Arf)) or Trp53 (encoding p53, a downstream target of p19(Arf)) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore, we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together, our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions, at least in part, via Ink4a/Arf and Trp53.

Unlabelled: AIMD: To assess the long-term effectiveness of an integrated disease management (IDM) program (consisting of optimal medication, reactivation, education, and exacerbation management) in primary care patients with chronic obstructive pulmonary disease (COPD)., Method: Controlled trial comparing the effects of IDM on quality of life--assessed by the St. George's Respiratory Questionnaire (SGRQ)--in primary care COPD patients. The minimal clinically important change on the SGRQ was accepted as being -4 points. Baseline and one year differences were compared using paired sample T-tests. The differential effects of an FEV1/FVC ratio <0.7 and dyspnoea as assessed by the Medical Research Council (MRC) Dyspnoea scale were investigated., Results: The average age of subjects was 63 years, with an average post-bronchodilator FEV1 of 67% predicted, average FEV1/FVC ratio of 0.65, a mean of 35 pack-years smoking, and 63% were male. No significant differences existed between groups at baseline. After one year of IDM, SGRQ had improved by -4.6 points (95% CI, -7.2 to -2.0; p=0.001) in the intervention group, versus -0.7 points (95% CI, - 3.0 to 1.6; p=0.6) in the usual care group. In patients with an FEV1/FVC ratio <0.7, SGRQ improved by -5.9 points (95% CI, -9.6 to -2.2; p=0.002) in the IDM group, while in the usual care group SGRQ improved by -0.8 points (95% CI, -4.1 to 2.4; p=0.6). In patients with an MRC Dyspnoea score >2 and FEV1/FVC <0.7, SGRQ improved by -13.4 points (95% CI, -20.8 to -6.1; p=0.002) in the IDM group, versus -0.3 points (95% CI, -5.5 to 4.9; p=0.9) in the usual care group., Conclusion: In this study, IDM improved one-year quality of life in primary care COPD patients, compared to usual care. The improvement in SGRQ was both clinically relevant and statistically significant, and was greatest in patients with FEV1/FVC <0.7 and MRC Dyspnoea score >2.

Recent developments in stem cell research have enabled the reprogramming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, and are being used to elucidate the molecular events that permit the conversion of one cell type to another. iPS cells have potential uses in in vitro disease modeling and toxicology screening, and as cellular therapies and regenerative medicine; however, various safety concerns exist that must be resolved before iPS cell therapy becomes a reality. Potential risks are related to the delivery of the endogenous factors, alterations in target cells, the cellular effects of the expression and reactivation of the factors that induce pluripotency, and safety issues related to the incorrect characterization and incomplete differentiation of the reprogrammed cells. In this review, the technique used to generate iPS cells is described, followed by a discussion of the safety concerns and how these concerns are currently being addressed.

With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID xenotransplantation model is still the favored model of choice in most cases, this system has some limitations as well, such as its cost-effectiveness, duration, and the lack of engraftability of cells from subsets of acute myeloid leukemia (AML) patients. Here, we have described an ex vivo bone marrow stromal coculture system in which CD34(+) cells, but not CD34(-) cells, from the bone marrow or peripheral blood of AML patients can give rise to long-term cultures (LTC) that can be maintained for over 20 weeks. Long-term expansion is associated with the formation of leukemic cobblestone area (L-CA) formation underneath the stroma. Self-renewal within these L-CAs can be determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which results in the generation of second, third, and fourth L-CAs that are able to sustain long-term expansion and generate high numbers of immature undifferentiated suspension cells. Furthermore, we have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in AML CD34(+) cells, and this method has also been described here. Together, these tools should allow a further molecular elucidation of derailed signal transduction in AML stem cells.

Objective: Mucin1 is a membrane glycoprotein that is overexpressed in a variety of human cancers. Here, we analyzed the role of Mucin1 in human hematopoietic stem/progenitor cells as well as in acute myeloid leukemia (AML) cells., Materials and Methods: Mucin1 expression was determined within the normal stem cell and progenitor compartment, as well as in the AML CD34+ and CD34- subfractions of patient samples. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays in limiting dilution and progenitor frequencies in colony-forming cell (CFC) assays in methylcellulose, and consequences of elevated Mucin1 expression were studied using retroviral overexpression systems in cord blood (CB) CD34+ cells., Results: Ten percent of CB and 5% of peripheral blood CD34+ cells expressed Mucin1. Retroviral overexpression of Mucin1 in CB CD34+ cells resulted in elevated stem cell and progenitor frequencies as determined in LTC-IC and CFC assays without affecting differentiation, which coincided with increased proliferation. Overexpression of intercellular adhesion molecule-1, a ligand for Mucin1, in MS5 stromal cells further increased LTC-IC frequencies. Mucin1 overexpression was associated with increased nuclear factor-kappaB p50 nuclear translocation, suggesting that Mucin1-induced phenotypes involve increased cell survival mechanisms. Finally, we observed increased Mucin1 expression in 70% of the AML cases (n=24), suggesting that elevated Mucin1 levels might be involved in regulating the proliferative potential of the immature leukemic compartment as well., Conclusions: Our data indicate that hematopoietic stem cells as well as CD34+ AML subfractions are enriched for Mucin1 expression, and that overexpression of Mucin1 in CB cells is sufficient to increase both progenitor and LTC-IC frequencies.

The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34+ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34+/CD38(-) cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34+ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% +/- 17%) and a decreased expansion (17 +/- 15 vs 80 +/- 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.

Objective: With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology., Methods: We have cultured acute myeloid leukemia CD34(+) cells on bone marrow stroma. Long-term expansion was monitored and self-renewal was addressed by replating of Leukemic-cobblestone area-forming cells (L-CAs). Also, lentiviral vectors were generated that could target L-CAs., Results: A strong expansion was observed in about 75% of the acute myeloid leukemia cases (n = 30) and long-term cultures could be maintained for up to 24 weeks on MS5 bone marrow stromal cells. Cells that were able to initiate leukemic cobblestone areas resided in the CD34(+) population and were absent from the CD34(-) population. Self-renewal within these L-CAs was determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which resulted in the generation of second, third, and fourth L-CAs, which were able to sustain long-term expansion and generated high numbers of immature undifferentiated suspension cells. CD34(+) cells that were able to initiate long-term cultures all coexpressed MEIS1 and HOXA9, and expressed elevated BMI1 levels., Conclusion: We present a novel long-term leukemic stem/progenitor assay in which new drugs can be tested and in which genes can be overexpressed or downmodulated using a lentiviral approach in order to obtain more insight into the process of leukemic transformation and self-renewal.

The CCAAT/enhancer binding protein (C/EBP) alpha transcription factor is indispensable for myeloid differentiation. In various myeloid leukemias, C/EBPalpha is mutated or functionally impaired due to decreased C/EBPalpha expression or phosphorylation. In order to investigate the functional consequences of decreased C/EBPalpha function in AML, we reintroduced C/EBPalpha in primary CD34(+) sorted acute myeloid leukemia (AML) cells using a lentiviral approach. Self-renewal and differentiation of primary AML stem cells were studied on long-term MS5 cocultures. Activation of C/EBPalpha immediately led to a growth arrest in all AML cultures (N = 7), resulting in severely reduced expansion compared with control cultures. This growth arrest corresponded with enhanced myeloid differentiation as assessed by fluorescence-activated cell sorter (FACS) analysis for CD14, CD15, and CD11b. Myeloid differentiation was further confirmed by the up-regulation of neutrophil elastase and granulocyte colony-stimulating factor (G-CSF) receptor in C/EBPalpha transduced cells. C/EBPalpha-expressing AML CD34(+) cells failed to generate second and third leukemic cobblestone areas (L-CAs) in serial replating experiments, while control cultures could be sequentially passaged for more than 4 times, indicating that reintroduction of C/EBPalpha impaired the self-renewal capacity of the leukemic CD34(+) compartment. Together, our data indicate that low C/EBPalpha levels are necessary to maintain self-renewal and the immature character of AML stem cells.

Ground reaction force (GRF) measurement is important in the analysis of human body movements. The main drawback of the existing measurement systems is the restriction to a laboratory environment. This paper proposes an ambulatory system for assessing the dynamics of ankle and foot, which integrates the measurement of the GRF with the measurement of human body movement. The GRF and the center of pressure (CoP) are measured using two six-degrees-of-freedom force sensors mounted beneath the shoe. The movement of foot and lower leg is measured using three miniature inertial sensors, two rigidly attached to the shoe and one on the lower leg. The proposed system is validated using a force plate and an optical position measurement system as a reference. The results show good correspondence between both measurement systems, except for the ankle power estimation. The root mean square (RMS) difference of the magnitude of the GRF over 10 evaluated trials was (0.012 +/- 0.001) N/N (mean +/- standard deviation), being (1.1 +/- 0.1)% of the maximal GRF magnitude. It should be noted that the forces, moments, and powers are normalized with respect to body weight. The CoP estimation using both methods shows good correspondence, as indicated by the RMS difference of (5.1 +/- 0.7) mm, corresponding to (1.7 +/- 0.3)% of the length of the shoe. The RMS difference between the magnitudes of the heel position estimates was calculated as (18 +/- 6) mm, being (1.4 +/- 0.5)% of the maximal magnitude. The ankle moment RMS difference was (0.004 +/- 0.001) Nm/N, being (2.3 +/- 0.5)% of the maximal magnitude. Finally, the RMS difference of the estimated power at the ankle was (0.02 +/- 0.005) W/N, being (14 +/- 5)% of the maximal power. This power difference is caused by an inaccurate estimation of the angular velocities using the optical reference measurement system, which is due to considering the foot as a single segment. The ambulatory system considers separate heel and forefoot segments, thus allowing an additional foot moment and power to be estimated. Based on the results of this research, it is concluded that the combination of the instrumented shoe and inertial sensing is a promising tool for the assessment of the dynamics of foot and ankle in an ambulatory setting.

Background: Activating mutations in the RET gene, which encodes a tyrosine kinase receptor, often cause medullary thyroid carcinoma (MTC). Surgical resection is the only curative treatment; no effective systemic treatment is available. We evaluated imatinib, a tyrosine kinase inhibitor currently used to treat chronic myelogenous leukemia and gastrointestinal stromal tumors, as a potential drug for systemic treatment of MTC, in 2 MTC-derived cell lines expressing multiple endocrine neoplasia-associated mutant RET receptors., Methods: We determined RET expression and Y1062 phosphorylation using Western blot analysis and quantitative polymerase chain reaction. We determined the effects on cell proliferation by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and we used fluorescence-activated cell sorter analysis with annexin V/propidium iodide staining to study imatinib-induced cell-cycle arrest, apoptosis, and cell death., Results: Imatinib inhibited RET Y1062 phosphorylation in a dose-dependent manner after 1.5 hours of exposure. After 16 hours both RET Y1062 phosphorylation and protein expression levels were affected. Dose-dependent decreases in cell proliferation of both cell lines after exposure to imatinib with inhibitory concentration of 50% levels of 23 +/- 2 micromol/L and 25 +/- 4 micromol/L were seen. These values are high, compared with those for chronic myelogenous leukemia and gastrointestinal stromal tumors. We further could show that imatinib induced cell-cycle arrest, and apoptotic and nonapoptotic cell death., Conclusions: Imatinib inhibits RET-mediated MTC cell growth affecting RET protein levels in vitro in a dose-dependent manner. The concentration of imatinib necessary to inhibit RET in vitro, however, makes it impossible to conclude that imatinib monotherapy will be a good option for systemic therapy of MTC.

Previously, we demonstrated that enforced activation of signal transducer and activator of transcription 5 (STAT5A) in human cord blood (CB)-derived stem/progenitor cells results in enhanced self-renewal and impaired myelopoiesis. The present study identifies C/EBPalpha as a critical component that is down-regulated by STAT5. Microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on STAT5A(1*6)-transduced CD34(+) cells identified C/EBPalpha as the most prominently down-regulated gene. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPalpha-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34(+) cells using a retroviral approach. Re-expression of C/EBPalpha in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis. The proliferative advantage imposed on CD34(+) cells by STAT5A(1*6) depended on the down-modulation of C/EBPalpha, as reintroduction of C/EBPalpha induced a quick cell-cycle arrest and the onset of myeloid differentiation. Long-term culture-initiating cell (LTC-IC) frequencies were elevated from 0.8% +/- 0.6% to 7.8% +/- 1.9% by STAT5A(1*6) as compared with controls, but these elevated LTC-IC frequencies were strongly reduced upon re-introduction of C/EBPalpha in STAT5A(1*6) cells, and no second cobble-stone area-forming cells (CAFCs) could be generated from double-transduced cells. Enumeration of progenitors revealed that the number of colony-forming cells (CFCs) was reduced more than 20-fold when C/EBPalpha was co-expressed in STAT5A(1*6) cells. Our data indicate that down-modulation of C/EBPalpha is a prerequisite for STAT5-induced effects on self-renewal and myelopoiesis.

Objective: To examine whether oncogenic Ras affects transforming growth factor (TGF)-beta-mediated cell-cycle arrest in hematopoietic cells and the downstream signal transduction pathway involved in the interference with TGF-beta-induced cell-cycle arrest., Materials and Methods: Two leukemic cell lines bearing N-Ras(L61) mutations; HL-60 and TF-1, and the M1 cell line with wt Ras were investigated for their response to TGF-beta. Signal transduction inhibitors, overexpression and RNA interference studies were performed to investigate the involvement of the various proteins., Results: Although TGF-beta signal transduction was not affected, G0-G1 arrest was absent in HL-60 and TF-1 cells due to the absence of p27. Overexpression of p27 restored TGF-beta-induced cell-cycle arrest, as well as interfering in Ras-mediated signaling. The farnesyl transferase inhibitor L744832 and the MEK inhibitor U0126 both restored p27 levels and cell-cycle arrest in response to TGF-beta. The absence of p27 protein is due to elevated levels of the ubiquitin ligase SKP2, which complexes with and targets p27 for degradation. RNA interference for SKP2 and treatment of these cells with the proteasome inhibitor MG132 restored p27 levels, corresponding with decreasing SKP2 levels after interfering in N-Ras signal transduction. P27, phosphorylated at threonine 187, is nuclear localized in N-Ras-containing cells. Mutation of this residue to alanine rendered p27 insensitive to degradation., Conclusion: N-Ras(L61) transformed cells lack a G0-G1 arrest upon TGF-beta treatment due to absence of p27. p27 is degraded through a MapK-, and SKP2-dependent pathway. Overexpression of p27 results in restoration of cell-cycle arrest upon TGF-beta treatment.

Objective: To investigate 1) the signal transduction pathways affected by heat shock protein 27 (HSP27) expression; and 2) the expression and regulation of HSP27 in acute myeloid leukemia (AML)., Materials and Methods: RNA interference studies for HSP27 in leukemic TF-1 cells were used to investigate the effects on downstream signal transduction and apoptosis after VP-16 and CD95/Fas treatment. HSP27 expression and activation was investigated in AML blasts through Western blot analysis., Results: RNA interference for HSP27 resulted in a twofold increase in VP-16-induced apoptosis, which was preceded by enhanced p38 and c-Jun phosphorylation and a twofold increased cytochrome c release into the cytoplasm. DAXX co-immunoprecipitated with HSP27, suggesting an inhibitory role of HSP27 in VP-16-mediated activation of the ASK1/p38/JNK pathway. CD95/Fas-induced apoptosis, however, was unaffected by HSP27 siRNA, due to upregulation of HSP27. Although HSP27 was highly expressed and phosphorylated in primitive monocytic AML blasts (M4-M5, 91%, n=11) and undetectable in myeloid blasts (M1-M2, n=5), VP-16-mediated apoptosis correlated moderately with HSP27 expression. This is likely due to the co-expression of p21Waf1/Cip1, which is in the majority of the monocytic AML M4-M5 blasts constitutively localized in the cytoplasm. Overexpression of cytoplasmic p21 inhibited the enhanced p38 phosphorylation after HSP27 RNAi, suggesting a predominant anti-apoptotic role of p21 over HSP27., Conclusion: 1) HSP27 inhibits VP-16-mediated phosphorylation of p38 and c-Jun, cytochrome c release, and subsequent apoptosis; 2) HSP27 is expressed and activated in monocytic AML blasts; 3) cytoplasmic expression of p21 compensates for the lack of HSP27.

In the present study, we analysed the expression and localization of p21(Waf1/Cip1) in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-kappaB (NF-kappaB) independent, results in predominantly cytoplasmic localized p21 protein. In acute monocytic leukaemia (M4, M5), monocytic blasts (N=12) show constitutive cytoplasmic p21 expression in 75% of the cases, while in myeloid leukaemic blasts (N=10), low nuclear and cytoplasmic localization of p21 could be detected, which is also PKC dependent. Constitutive p21 expression in monocytic leukaemia might have important antiapoptotic functions. This is supported by the finding that in U937 cells overexpressing p21, VP16-induced apoptosis is significantly reduced (20.0+/-0.9 vs 55.8+/-3.8%, P<0.01, N=5), reflected by a reduced phosphorylation of p38 and JNK. Similarly, AML blasts with high cytoplasmic p21 were less sensitive to VP16-induced apoptosis as compared to AML cases with low or undetectable p21 expression (42.25 vs 12.3%, P<0.01). Moreover, complex formation between p21 and ASK1 could be demonstrated in AML cells, by means of coimmunoprecipitation. In summary, these results indicate that p21 has an antiapoptotic role in monocytic leukaemia, and that p21 expression is regulated in a PKC-dependent and NF-kappaB-independent manner.

Activation of the signal transducer and activator of transcription 3 (STAT3) in response to interleukin-6 (IL-6) type cytokines involves both phosphorylation of Tyr705, which enables dimerization, nuclear translocation and DNA binding, as well as ser727 phosphorylation. Here, we describe that the 65 C-terminal amino acids of STAT3 can function as an independent transcription activation domain (TAD), particularly when a negative charge is introduced at position 727 by mutation of the serine residue into aspartate. The strong transcriptional activity of the C-terminal STAT3 Ser727Asp TAD is coupled to a constitutive association with the co-activator p300. In HepG2 cells, p300 associates with STAT3 upon IL-6 stimulation, and overexpression of p300 enhances the transcriptional activity of STAT3alpha, but not of STAT3beta or STAT3 Ser727Ala. We conclude that Ser727 phosphorylation in the C-terminal region of STAT3 is required for transactivation by association with p300.

We report here about the relationship between the destabilization of the lipid organization induced by praziquantel and derivatives and their mode of insertion into the lipid matrix. Measurements of lipid transition temperature and efflux of 6-carboxyfluorescein encapsulated in liposomes establish the lipid destabilizing capacity of praziquantel as compared to praziquantel derivatives. IR spectroscopy (attenuated total reflection technique) applied to oriented lipid bilayers indicates that praziquantel or derivatives do not modify significantly the lipid structure. In order to give a molecular description of the position of the drug into the lipid bilayer, we applied a conformational analysis procedure making it possible to calculate the structure of amphiphilic molecules assembled in aggregates and the mode of insertion of amphiphilic drugs into a lipid layer. The praziquantel lipid destabilizing capacity is explained in terms of the high praziquantel-lipid interaction and the large area occupied per drug molecule in the lipid layer.