Functional characterisation of a novel nucleoporin gene NUP98 in zebrafish embryo.

Oral PresentationINTRODUCTION: The nucleoporin gene nup98 is important for the regulation of cytoplasmic-nuclear trafficking. Frequent disruptions of NUP98 during chromosomal translocation in acute myeloid leukaemia suggest that it may play a role in normal haematopoiesis. nup98-knockout mice has resulted in early embryonic lethality. Therefore, its role in embryonic haematopoiesis remains unclear. In this study, we have cloned a zebrafish nup98 gene and examined its role in embryonic development, with particular reference to haematopoiesis. METHODS: Two expressed sequence tags with translated sequence homologous to human NUP98 were identified. The gene was cloned by PCR from cDNA of zebrafish embryos. Expression of nup98 in zebrafish embryos was investigated spatially by whole-mount in-situ hybridisation and temporally by RT-PCR. The functions of nup98 were examined by morpholino knockdown and the effects on embryonic development evaluated by gene expression studies and confocal microscopy. Cellular functions of zebrafish nup98 were investigated in HeLa cells. RESULTS: Zebrafish nup98 gene shared 65% identity to human NUP98 homolog in protein sequence. The gene was expressed during early embryonic development since 1-cell stage and diffusely in eyes and the developing brain since 18 hpf. About 30% nup98-knockdown embryos developed intracranial haemorrhage at 48 hpf, resulting from disrupted blood vessels. nup98-knockdown upregulated pu.1 and scl as evaluated by quantitative RT-PCR. Moreover, ectopic expression of zebrafish nup98 rescued the defective mRNA export due to NUP98 knockdown in HeLa cells. CONCLUSION: A novel zebrafish nup98 gene was shown to exhibit conserved function in mRNA trafficking. Its role in embryonic development should be further evaluated.published_or_final_versio

Methylation of cyclin-dependent kinase inhibitors, XAF1, JUNB, CDH13 and soluble Wnt inhibitors in essential thrombocythaemia

Background: Methylation of genes regulating cell-cycle check-point (INK4 cyclin-dependent kinase inhibitors), apoptosis (XAF1), adhesion (CDH13), JUNB and Wnt signalling (soluble Wnt inhibitors) has been implicated in pathogenesis of haematological and epithelial cancers. Method The authors studied the methylation status of CDKN2A, CDKN2B, XAF1, CDH13, JUNB and a panel of soluble Wnt inhibitors including WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 and SFRP5 by methylation-specific PCR in 31 bone marrow and 21 peripheral blood samples of patients with essential thrombocythaemia. Results and discussion: There was no evidence of hypermethylation of all these genes in both the BM and PB samples. Therefore, in contrast to myeloid leukaemias, methylation of these genes regulating the cell cycle, apoptosis, adhesion and Wnt signalling does not play an important role in the pathogenesis of myeloproliferative diseases. Whether differential methylation may occur in the progenitor or mature blood cell compartments remains to be verified. Our study contributes to the literature on methylation in chronic myeloproliferatve diseases.published_or_final_versio

Methylation of TET2, CBL and CEBPA in Ph-negative myeloproliferative neoplasms

A loss-of-function mutation of TET2, CBL and CEBPA has been implicated in the pathogenesis or leukaemic transformation of myeloproliferative neoplasm. As tumour suppressor genes may potentially be inactivated by promoter hypermethylation, the authors studied the methylation status of these genes in three cell lines and diagnostic marrow samples from 45 patients with myeloproliferative neoplasm (MPN) (essential thrombocythaemia, N=34; polycythaemia vera, N=7 and primary myelofibrosis, N=4) by methylation-specific PCR. TET2 was heterozygously methylated in MEG-01 and K562 but completely unmethylated in HEL. On the other hand, both CBL and CEBPA were completely unmethylated in all three cell lines. In the primary marrow samples, methylation of TET2 occurred in two (5.9%) patients with essential thrombocythaemia (4.4% of all patients), both without JAK2 V617 mutation, but not in polycythaemia vera or primary myelofibrosis. There was no association between TET2 methylation with the type of MPN (p=0.713). Hypermethylation of CBL or CEBPA was not detected in any patients. In summary, methylation of TET2, CBL and CEBPA is infrequent in MPN at diagnosis. The role of methylation of these genes at the time of leukaemic transformation warrants further study.published_or_final_versio

Methylation profiling of SOCS1, SOCS2, SOCS3, CISH and SHP1 in Philadelphia-negative myeloproliferative neoplasm

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Alterations of CD8+CD28- T cells in systemic lupus erythematosus and rheumatoid arthritis

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Differential NOD/SCID mouse engraftment of peripheral blood CD34 + cells and JAK2V617F clones from patients with myeloproliferative neoplasms

We evaluated the NOD/SCID engraftment of CD34 + cells from polycythemia vera (PV) and secondary polycythemia patients (SP) and the JAK2V617F clone before and after transplantation. Peripheral blood CD34 + cells were transplanted intra-femorally. In the injected BM, successful engraftment (>0.1%) occurred in 8/26 mice transplanted with CD34+ cells from 5/13 PV patients (median: 4.26%, range: 0.3-5.56%), in contrast to 0/14 mice from 9 SP patients (P=0.017). The engrafting PV cells were of multi-lineage. JAK2V617F/total JAK2 ratios decreased after transplantation (initial: 65.9% versus 6-week: 13.0%, P=0.001). Essential thrombocythemia (ET) BM cells also exhibited a similar decrease in JAK2V617F clone. The results suggested that events in addition to JAK2V617F are involved in the pathogenesis of PV and ET. © 2010 Elsevier Ltd.postprin

ACER eNews Issue 04 April 2013

Cell mechanics, in particular mechanical properties, has been suggested as a new biomarker indicative of cell state and phenotype. Acute myeloid leukemia (AML) is characterized by the abnormal increase of myeloblasts in blood and bone marrow. While AML has been extensively studied from the perspectives of biochemical and genetic aspects, little is known about its cellular biophysical properties. In this study, optical tweezer technology was used to examine the micromechanical properties of myeloblasts from bone marrow of AML patients at single cell level. The myeloblasts were separately analyzed according to their expression of CD34 +, a marker of primitive hematopoietic cells. To extract the intrinsic properties from the relationship between the stretching force and the induced deformation, a theoretical approach was developed to model the mechanical responses of cells and further characterize their mechanical properties. The preliminary results show that the area compressibility modulus of CD34 + myeloblasts was significantly less than that of CD34 - cells, which indicate that micromechanical properties are unique features of myeloblasts and provide us with an insight into the cell mechanics of primitive AML cells. © 2010 IEEE.published_or_final_versio

Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms

BACKGROUND: MicroRNA (miR) miR-34a, -34b/c, -124-1 and -203 are tumor suppressor miRs implicated in carcinogenesis. METHODS: We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs. RESULTS: Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation. CONCLUSION: This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.published_or_final_versio

Prevalence of occult hepatitis B infection in a highly endemic area for chronic hepatitis B: A study of a large blood donor population

Background and aims: The aim of the present study was to determine the population prevalence of occult hepatitis B (OHB) infection and its clinical profile in a highly endemic area of chronic hepatitis B virus disease. Methods: OHB was first identified by individual sample testing for hepatitis B surface antigen (HBsAg) followed by nucleic acid testing (NAT) and vice versa for 3044 (cohort 1, stored sera from donation within 1 year) and 9990 (cohort 2, prospective study) blood donors, respectively. OHB was confirmed meticulously by ≥2 out of 3 tests with detectable hepatitis B virus (HBV) DNA using a sensitive standardised assay. Detailed serology and viral load in the serum and liver were studied. Results: The prevalence of OHB was 0.13% (4/3044) and 0.11% (11/9967) for cohort 1 and 2, respectively. In cohort 2, 10 out of 11 OHB samples were positive for anti-HBc (hepatitis B core antigen) antibody (all were immunoglobulin G). Seven had detectable anti-HBs. The serum HBV DNA levels were extremely low (highest 14.1 IU/ml). Of the six donors who underwent liver biopsies, all had normal liver biochemistry, extremely low liver HBV DNA (highest 6.21 copies/cell) and nearly normal liver histology. For those with viral sequence generation, none had the common HBsAg mutant G145R. Conclusions: The prevalence of OHB in a highly endemic area of chronic HBV was very low, thus implying a low impact on transfusion services. To implement universal screening, the high cost of NAT should be taken into account. OHB blood donors had very low HBV replication, and normal liver biochemistry and histology, conferring a favourable prognosis.published_or_final_versio

Recalculation of 11‐year total ozone of Brewer spectrophotometer 115

Author name used in this publication: K. S. Lam2007-2008 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe