Modulation of p21 Human Tumor Suppressor Gene Expression By Zinc Status In Human Hepatoblastoma And Normal Human Bronchial Epithelial Cells

The transcriptional regulation of p21 gene in both human hepatoblastoma (HepG2) cells and normal human bronchial epithelial (NHBE) cells in response to different zinc status has been examined. In both zinc deficient (ZD) and zinc marginal deficient (ZD0.4) HepG2 cells, the p21 mRNA and nuclear protein levels as well as p21 promoter activity were repressed as compared to that of zinc normal (ZN) control cells. However, they were not altered in zinc adequate (ZA) and zinc supplement (ZS) cells as compared to ZN cells. Moreover, transfection of a construct consisted of a constitutive promoter fused with a full length p21 coding sequence in ZD cells, normalized p21 protein expression to that of the ZN cells, but failed to correct cell growth reduction. Similar transfection in ZN cells overexpressed p21 and repressed cell growth. Thus, the present data indicate that in zinc-deficient HepG2 cells, the p21 transcriptional process is depressed. However, depressed p21 transcriptional process is not responsible for repressed cell growth in zinc-deficient HepG2 cells. In NHBE cells, the nuclear p21 protein level and mRNA abundance as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost 2-fold when compared to ZN control cells. G2/M blockage in ZS cells was coupled with enhanced p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage. A similar gene knock-down approach was used to establish if the p21 upregulation in ZS cells was p53 dependent. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53-dependent. Furthermore, the normalization of p53 protein by siRNA in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status induced G2/M blockage and growth depression. Thus, high zinc status in NHBE cells upregulated p53 expression which in turn elevated p21 that eventually induced G2/M blockage

SETDB2 Links E2A-PBX1 to Cell-Cycle Dysregulation in Acute Leukemia through CDKN2C Repression

Acute lymphoblastic leukemia (ALL) is associated with significant morbidity and mortality, necessitating further improvements in diagnosis and therapy. Targeted therapies directed against chromatin regulators are emerging as promising approaches in preclinical studies and early clinical trials. Here, we demonstrate an oncogenic role for the protein lysine methyltransferase SETDB2 in leukemia pathogenesis. It is overexpressed in pre-BCR+ ALL and required for their maintenance in vitro and in vivo. SETDB2 expression is maintained as a direct target gene of the chimeric transcription factor E2A-PBX1 in a subset of ALL and suppresses expression of the cell-cycle inhibitor CDKN2C through histone H3K9 tri-methylation, thus establishing an oncogenic pathway subordinate to E2A-PBX1 that silences a major tumor suppressor in ALL. In contrast, SETDB2 was relatively dispensable for normal hematopoietic stem and progenitor cell proliferation. SETDB2 knockdown enhances sensitivity to kinase and chromatin inhibitors, providing a mechanistic rationale for targeting SETDB2 therapeutically in ALL

Restoring Hong Kong's landscar in the Anderson Quarry : an analysis of civic engagement strategies

published_or_final_versionPolitics and Public AdministrationMasterMaster of Public Administratio

Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation

In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms

Towards a global partnership model in interprofessional education for cross-sector problem-solving

Objectives A partnership model in interprofessional education (IPE) is important in promoting a sense of global citizenship while preparing students for cross-sector problem-solving. However, the literature remains scant in providing useful guidance for the development of an IPE programme co-implemented by external partners. In this pioneering study, we describe the processes of forging global partnerships in co-implementing IPE and evaluate the programme in light of the preliminary data available. Methods This study is generally quantitative. We collected data from a total of 747 health and social care students from four higher education institutions. We utilized a descriptive narrative format and a quantitative design to present our experiences of running IPE with external partners and performed independent t-tests and analysis of variance to examine pretest and posttest mean differences in students’ data. Results We identified factors in establishing a cross-institutional IPE programme. These factors include complementarity of expertise, mutual benefits, internet connectivity, interactivity of design, and time difference. We found significant pretest–posttest differences in students’ readiness for interprofessional learning (teamwork and collaboration, positive professional identity, roles, and responsibilities). We also found a significant decrease in students’ social interaction anxiety after the IPE simulation. Conclusions The narrative of our experiences described in this manuscript could be considered by higher education institutions seeking to forge meaningful external partnerships in their effort to establish interprofessional global health education

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The anatomical and temporal expression of N-methyl-D-aspartate receptor (NMDAR) subunit NMDAR3 (NR3) in the mammalian brain using a novel monoclonal anti-NR3 antibody

NR3 is a novel, developmentally regulated NMDA receptor (NMDAR) subunit that was previously known as NMDAR-Like (NMDAR-L) or χ-1 subunit. The discovery of NR3 suggested a role for this novel subunit in neurogenesis as expression of NR3 transcripts peaked at the end of the first postnatal week and was reduced to a low level in adulthood. The regulatory role in neural development was further supported by the evidence that mice in which NR3 was ablated by gene targeting showed increased NMDA-induced currents and increased density of dendritic spines in early postnatal cerebrocortical neurons. These data suggest an inhibitory role of NR3 in NMDAR functions and that it may be involved in the development of synaptic elements by modulating NMDAR activity. To date, the localization of NR3 at the protein level is still unknown. To examine the expression of NR3 protein in the brain is essential to explore the regulatory role of NR3 in the context of NMDAR functions. Therefore, a novel monoclonal anti-NR3 antibody was generated and characterized to delineate the anatomical and temporal expression of NR3 protein in the mammalian brain. The monoclonal anti-NR3 was found to be specific for NR3 in immunocytochemistry, immunoprecipitation and Western blotting. NR3 protein was found to peak at postnatal day 8 (P8), and to decrease gradually from P12 to adulthood in rat central nervous system. NR3 protein was heavily expressed in all areas of the isocortex, a substantial portion of nuclei in the amygdala, in selective cell layers and nuclei in the hippocampal formation, the thalamus, the brainstem, the cerebellar cortex and the spinal cord. The information provided in this study will be useful for the future research of NMDARs, by contributing to the understanding on the role of NR3 and the possible stoichiometry of NMDARs in certain neuronal circuits