Methionine aminopeptidase 2 is required for HSC initiation and proliferation.

In a chemical screening, we tested the antiangiogenic effects of fumagillin derivatives and identified fumagillin as an inhibitor of definitive hematopoiesis in zebrafish embryos. Fumagillin is known to target methionine aminopeptidase II (MetAP2), an enzyme whose function in hematopoiesis is unknown. We investigated the role of MetAP2 in hematopoiesis by using zebrafish embryo and human umbilical cord blood models. Zebrafish metap2 was expressed ubiquitously during early embryogenesis and later in the somitic region, the caudal hematopoietic tissue, and pronephric duct. metap2 was inhibited by morpholino and fumagillin treatment, resulting in increased mpo expression at 18 hours postfertilization and reduced c-myb expression along the ventral wall of dorsal aorta at 36 hours postfertilization. It also disrupted intersegmental vessels in Tg(fli1:gfp) embryos without affecting development of major axial vasculatures. Inhibition of MetAP2 in CB CD34(+) cells by fumagillin had no effect on overall clonogenic activity but significantly reduced their engraftment into immunodeficient nonobese diabetes/severe combined immunodeficiency mice. metap2 knock-down in zebrafish and inhibition by fumagillin in zebrafish and human CB CD34(+) cells inhibited Calmodulin Kinase II activity and induced ERK phosphorylation. This study demonstrated a hitherto-undescribed role of MetAP2 in definitive hematopoiesis and a possible link to noncanonical Wnt and ERK signaling.

Hong Kong-Macau Severe Hives and Angioedema Referral Pathway.

Background: Urticaria (defined as the presence of hives, angioedema, or both) can be caused by a variety of etiologies ranging from more common conditions such as chronic spontaneous urticaria (CSU) to rarer conditions such as hereditary angioedema (HAE). Specialist referral may be necessary in cases of severe urticaria or HAE, but access to specialist services remains limited in certain regions, such as the Greater Bay Area (GBA) of China. To address this, the Hong Kong-Macau Severe Hives and Angioedema Referral Pathway (SHARP) was initiated by the Hong Kong Institute of Allergy and Macau Society of Dermatology to promote multidisciplinary collaboration and regional exchange of expertise in the diagnosis and management of severe urticaria. Methods: A nominated task force of dermatologists and immunologists who manage patients with severe urticaria formulated the consensus statements (CS) using the Delphi method. The consensus was defined a priori as an agreement of ≥80%. Results: A total of 24 CS were formulated, including four statements on classifications and definitions, seven statements on diagnosis, and 13 statements on management and referral. The definitions for acute/chronic urticaria and severe CSU were stated. Unnecessary investigations and inappropriate medications were discouraged. The characteristics and recommended approach to suspected bradykinergic angioedema were specified. Stepwise treatment options using second-generation antihistamines, omalizumab, or cyclosporin for patients with CSU were addressed, and the importance of access to HAE-specific medications was emphasized. Furthermore, an integrated referral pathway for patients with severe hives and angioedema was constructed. Conclusion: The SHARP provides guidance for the management and specialist referral of patients with severe hives and angioedema in Hong Kong and Macau.

Comparative genomics of elastin: Sequence analysis of a highly repetitive protein.

Due to the low complexity associated with their sequences, uncovering the evolutionary and functional relationships in highly repetitive proteins such as elastin, spider silks, resilin and abductin represents a significant challenge. Using the polymeric extracellular protein elastin as a model system, we present a novel computational approach to the study of sequence, function and evolutionary relationships in repetitive proteins. To address the absence of accurate sequence annotation for repetitive proteins such as elastin, we have constructed a new database repository, ElastoDB (http://theileria.ccb.sickkids.ca/elastin), dedicated to the storage and retrieval of elastin sequence- and meta-data. To analyse their sequence relationships we have devised an innovative new method, based on the identification of overrepresented 'fuzzy' motifs. Applying this method to elastin sequences derived from mammals, chicken, Xenopus and zebrafish resulted in the identification of both highly conserved, and taxon and species specific motifs that likely represent important functional and/or structural elements. The relative spacing and organization of these elements suggest that exon duplication events have played an important role in the evolution of elastin. Clustering of similarity profiles generated for sets of exons and introns, revealed a pattern of putative duplication events involving exons 15-30 in mammalian and chicken elastins, exons 20-31 in both zebrafish elastins, exons 15-20 in fugu elastin and exons 35-50 in Xenopus elastin 1. The success of this approach for elastin offers a promising route to the elucidation of sequence, structure, function and evolutionary relationships for many other proteins with sequences of low complexity.

Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: Clues to the evolutionary history of elastins.

Tropoelastin is the monomeric form of elastin, a polymeric extracellular matrix protein responsible for properties of extensibility and elastic recoil in connective tissues of most vertebrates. As an approach to investigate how sequence and structural characteristics of tropoelastin assist in polymeric assembly and account for the elastomeric properties of this polymer, and to better understand the evolutionary history of elastin, we have identified and characterized tropoelastins from frog (Xenopus tropicalis) and zebrafish (Danio rerio), comparing these to their mammalian and avian counterparts. Unlike other species, two tropoelastin genes were expressed in zebrafish. All tropoelastins shared a predominant and characteristic alternating domain arrangement, as well as the fundamental crosslinking sequence motifs. However, zebrafish and frog tropoelastins had several unusual characteristics, including increased exon numbers and protein molecular weights, and decreased hydropathies. For all tropoelastins there was evidence of evolutionary expansion of the proteins by extensive replication of a hydrophobic-crosslinking exon pair. This was particularly apparent for zebrafish and frog tropoelastin genes, where remnants of sequence similarity were also seen in introns flanking the replicated exon pair. While overall alignment of mammalian, avian, frog and zebrafish tropoelastin sequences was not possible because of sequence variability, the C-terminal exon was well-conserved in all species. In addition, good sequence alignment was possible for several exons just upstream of the putative region of replication, suggesting that these conserved domains may represent 'primordial' core sequences present in the ancestral sequence common to all tropoelastins and in some way essential to the structure/function of elastin.

Characterization of Sry-related HMG box group F genes in zebrafish hematopoiesis.

OBJECTIVE: The roles of Sry-related HMG box (Sox) genes in zebrafish hematopoiesis are not clearly defined. In this study, we have characterized the sequence homology, gene expression, hematopoietic functions, and regulation of sox genes in F group (SoxF) in zebrafish embryos. MATERIALS AND METHODS: Expression of zebrafish SoxF genes were analyzed by whole-mount in situ hybridization, reverse transcription polymerase chain reaction, and real-time reverse transcription polymerase chain reaction of erythroid cells obtained from Tg(gata1:GFP) embryos by fluorescence-activated cell sorting. Roles of SoxF genes were analyzed in zebrafish embryos using morpholino knockdown and analyzed by whole-mount in situ hybridization and real-time reverse transcription polymerase chain reaction. Embryo patterning and vascular development were analyzed. RESULTS: All members, except sox17, contained a putative ß-catenin binding site. sox7 and 18 expressed primarily in the vasculature. sox17 expressed in the intermediate cell mass and its knockdown significantly reduced primitive erythropoiesis at 18 hours post-fertilization (hpf). Definitive hematopoiesis was unaffected. Concomitant sox7 and sox18 knockdown disrupted vasculogenesis and angiogenesis, but not hematopoiesis. sox32 knockdown delayed medial migration of hematopoietic and endothelial progenitors at 18 hpf and abolished cmyb expression at the caudal hematopoietic tissue at 48 hpf. These defects could be prevented by delaying its knockdown using a caged sox32 morpholino uncaged at 10 hpf. Knockdown of SoxF genes significantly upregulated their own expression and that of sox32 also upregulated sox18 expression. CONCLUSIONS: sox17 helped to maintain primitive hematopoiesis, whereas sox7 and sox18 regulated angiogenesis and vasculogenesis. sox32 affected both vascular and hematopoietic development through its effects on medial migration of the hematopoietic and endothelial progenitors.

Role of a novel zebrafish nup98 during embryonic development.

OBJECTIVE: The nucleoporin NUP98 is a component of the nuclear pore complex that regulates nucleocytoplasmic trafficking. It has been characterized in acute myeloid leukemia as a fusion partner during chromosomal translocation. In this study, we identified a zebrafish nup98 gene and examined its role in embryonic development. MATERIALS AND METHODS: Two expressed sequence tags with translated sequences homologous to human NUP98 were identified. The gene was cloned by polymerase chain reaction from complementary DNA of zebrafish embryos. Cellular functions of zebrafish NUP98 were investigated in HeLa cells. nup98 expression and developmental functions in zebrafish embryos were investigated by whole-mount in situ hybridization and morpholino knockdown. RESULTS: Protein sequence of zebrafish nup98 shared 65% identity with its human homolog. Ectopic expression of zebrafish nup98 rescued the defective messenger RNA export due to human NUP98 knockdown in HeLa cells. In zebrafish embryos, nup98 was expressed diffusely in eyes and the developing brain since 18 hours postfertilization. Knockdown of nup98 with morpholino upregulated pu.1 expression by 39% ± 15% (p = 0.0153) and scl expression by 36% ± 7.6% (p = 0.0017). Expression of genes associated with erythropoiesis was unchanged. The morphants also developed intracranial hemorrhage at 48 hours postfertilization due to defective blood vessel development. CONCLUSIONS: A novel zebrafish nup98 was identified and it serves a role in nucleocytoplasmic trafficking similar to human NUP98. During development, it modulates hematopoietic stem cell and early myeloid development and maintains the integrity of cranial vasculature in the developing central nervous system.

Worksite health promotion: the value of the Tune Up Your Heart program.

Successful wellness initiatives at DaimlerChrysler Canada Incorporated (DCCI) led to a unique partnership between key stakeholders that allowed implementation of Tune Up Your Heart, a program aimed at improving workforce cardiovascular disease (CVD) risk. Volunteers were screened and stratified according to their CVD risk. Interventions were tailored to risk level and included goal setting, monitoring progress, and company-wide education programs. Outcome data (CVD risk and components of risk) were collected at study entry and after 18 months. The economic impact of the program was determined using a model based on subject movement across risk categories and historical claims data for life insurance, short- and long-term disability, prescription drugs, and casual absenteeism. Intervention participants (N = 343) demonstrated a significant (P = .0113) relative CVD risk reduction of 12.7%; 36% of participants lost weight, and average body mass index decreased from 28.4 to 28.2 (P = .0419). Average systolic and diastolic blood pressure significantly decreased (P < .0001 and P = .0221, respectively). Subjects reported increased adherence to recommended exercise and diet regimens, and the number of smokers decreased by 14%. The majority of subjects reported satisfaction with the program. Annual savings were estimated at Can$793 for the intervention group and Can$18,461 when projected to the entire workforce (N = 13,629). Savings were sensitive to cost weighting when subjects moved to a lower risk class but more robust to other parameters. The Tune Up Your Heart program significantly improved employee CVD risk profile, and was associated with savings for DCCI.