Phosphorylation State-Dependent Interactions of Hepadnavirus Core Protein with Host Factors

Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions

Evaluation of DNA ploidy in relation with established prognostic factors in patients with locally advanced (unresectable) or metastatic pancreatic adenocarcinoma: a retrospective analysis

<p>Abstract</p> <p>Background</p> <p>Most patients with ductal pancreatic adenocarcinoma are diagnosed with locally advanced (unresectable) or metastatic disease. The aim of this study was to evaluate the prognostic significance of DNA ploidy in relation with established clinical and laboratory variables in such patients.</p> <p>Methods</p> <p>Two hundred and twenty six patients were studied retrospectively. Twenty two potential prognostic variables (demographics, clinical parameters, biochemical markers, treatment modality) were examined.</p> <p>Results</p> <p>Mean survival time was 38.41 weeks (95% c.i.: 33.17–43.65), median survival 27.00 weeks (95% c.i.: 23.18–30.82). On multivariate analysis, 10 factors had an independent effect on survival: performance status, local extension of tumor, distant metastases, ploidy score, anemia under epoetin therapy, weight loss, pain, steatorrhoea, CEA, and palliative surgery and chemotherapy. Patients managed with palliative surgery and chemotherapy had 6.7 times lower probability of death in comparison with patients without any treatment. Patients with ploidy score > 3.6 had 5.0 times higher probability of death in comparison with patients with ploidy score < 2.2 and these with ploidy score 2.2–3.6 had 6.3 times higher probability of death in comparison with patients with ploidy score < 2.2.</p> <p>Conclusion</p> <p>According to the significance of the examined factor, survival was improved mainly by the combination of surgery and chemotherapy, and the presence of low DNA ploidy score.</p

Differential expression of glutamate decarboxylase isoforms in the horizontal cells of human and rat retinas

L-glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, catalyze thesynthesis of c-aminobutyric acid (GABA). Previous studies demonstrated thatGAD65 and GAD67 appear only in the horizontal cells of monkey, and rodentsand cats, respectively; whereas both GAD65 and GAD67 can be found in therabbit horizontal cells. In the present study, we used immunohistochemistry andWestern blot to examine the expression of GAD isoforms in the retinas of humanfetuses and also in the developing and adult rat retinas. We found that both GADisoforms existed in the amacrine cells and the inner plexiform layer of both humanand rat retinas. GAD67 was found in the more early stage and more in the cellbodies. Two GAD isoforms were detected in a different stratification pattern of theinner plexiform layer. However, only GAD65 was observed transiently in thehorizontal cells, at 22 WG of human fetus; while both isoforms were found in rats.GAD67 was first seen at birth and GAD65 at postnatal 14 days and through adultin rats. Previous studies provided evidences that GABAc receptor is expressed inthe bipolar cells and cone photoreceptors. Thus GABA released by horizontal cellswas hypothesized to act as a forward feedback to bipolar cells and a backwardfeedback to cone photoreceptors. In summary, GAD isoforms catalyzing GABAsynthesis in the horizontal cells are different among different species and onlyGAD65 plays a role in the horizontal cells of human fetuses.Acknowledgements: Support: The University of Hong Kong, Institute ofMolecular Technology for Drug Discovery and Synthesis of HKU, and ResearchGrant Council of Hong Kong

Differential expression of glutamate decarboxylase isoforms in the horizontal cells of human and rat retinas

L-glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, catalyze thesynthesis of c-aminobutyric acid (GABA). Previous studies demonstrated thatGAD65 and GAD67 appear only in the horizontal cells of monkey, and rodentsand cats, respectively; whereas both GAD65 and GAD67 can be found in therabbit horizontal cells. In the present study, we used immunohistochemistry andWestern blot to examine the expression of GAD isoforms in the retinas of humanfetuses and also in the developing and adult rat retinas. We found that both GADisoforms existed in the amacrine cells and the inner plexiform layer of both humanand rat retinas. GAD67 was found in the more early stage and more in the cellbodies. Two GAD isoforms were detected in a different stratification pattern of theinner plexiform layer. However, only GAD65 was observed transiently in thehorizontal cells, at 22 WG of human fetus; while both isoforms were found in rats.GAD67 was first seen at birth and GAD65 at postnatal 14 days and through adultin rats. Previous studies provided evidences that GABAc receptor is expressed inthe bipolar cells and cone photoreceptors. Thus GABA released by horizontal cellswas hypothesized to act as a forward feedback to bipolar cells and a backwardfeedback to cone photoreceptors. In summary, GAD isoforms catalyzing GABAsynthesis in the horizontal cells are different among different species and onlyGAD65 plays a role in the horizontal cells of human fetuses.Acknowledgements: Support: The University of Hong Kong, Institute ofMolecular Technology for Drug Discovery and Synthesis of HKU, and ResearchGrant Council of Hong Kong

Flk-1, a receptor for vascular endothelial growth factor, is expressed in the neural cells of developmental human retinas

Presentation no. 453.12Membrane receptor tyrosine kinase Flk-1 (also designated VEGF-R2) is a high-affinity receptor for vascular endothelial growth factor (VEGF) and putatively involved in the growth of the vascualr endothelial cells. However, recently some researchers found that Flk-1 expresses in retinal progenitor cells of developmental mouse retinas and a role of VEGF for neural retinal development is implied. In the present study, we investigated the expression of Flk-1 and VEGF in the developmental human retinas ranging from 10.5 weeks of gestation (WG) to 22 WG by immunohistochemistry. We found that as early as 10.5 WG, Flk-1-immunoreactivity (IR) expresses in cell membrane of retinal progenitor cells of the inner retinas. At 17 WG, Flk-1-IR is expressed strongly in many cells of the ganglion cell layer and weakly in some cells at the innermost of inner nuclear layer. After 21 WG, Flk-1-IR is still expressed in cells of ganglion cell layer and most strongly in cells at innermost of inner nuclear layer and in addition, in horizontal cells. Flk-1-IR also expresses in choroid vessels outside the retinas at all stages, and in the retinal vessels after 18 WG. Meanwhile, VEGF-IR occurs in the Muller cells through all these time periods. In conclusion, we have shown the expression of Flk-1 in retinal progenitor cells and later on in neural retinal cells before retinal vessels occur in the retinas. Together with the consistent expression of VEGF in this developmental period, this study suggests that VEGF plays a nonvascular role for the development of human retinas. Supported by The University of Hong Kong, Research Grant Council of Hong Kong and Croucher Foundatio

Expression of glutamate decarboxylase isoforms in human and rat retinas

L-glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, catalyze thesynthesis of c-aminobutyric acid (GABA). Previous studies demonstrated thatGAD65 and GAD67 appear only in the horizontal cells of monkey, and rodentsand cats, respectively; whereas both GAD65 and GAD67 can be found in therabbit horizontal cells. In the present study, we used immunohistochemistry andWestern blot to examine the expression of GAD isoforms in the retinas of humanfetuses and also in the developing and adult rat retinas. We found that both GADisoforms existed in the amacrine cells and the inner plexiform layer of both humanand rat retinas. GAD67 was found in the more early stage and more in the cellbodies. Two GAD isoforms were detected in a different stratification pattern of theinner plexiform layer. However, only GAD65 was observed transiently in thehorizontal cells, at 22 WG of human fetus; while both isoforms were found in rats.GAD67 was first seen at birth and GAD65 at postnatal 14 days and through adultin rats. Previous studies provided evidences that GABAc receptor is expressed inthe bipolar cells and cone photoreceptors. Thus GABA released by horizontal cellswas hypothesized to act as a forward feedback to bipolar cells and a backwardfeedback to cone photoreceptors. In summary, GAD isoforms catalyzing GABAsynthesis in the horizontal cells are different among different species and onlyGAD65 plays a role in the horizontal cells of human fetuses.Acknowledgements: Support: The University of Hong Kong, Institute ofMolecular Technology for Drug Discovery and Synthesis of HKU, and ResearchGrant Council of Hong Kong

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

Fosfomycin-based combination therapy has emerged as an attractive option in our armamentarium due to its synergistic activity against carbapenem-resistant Gram-negative bacteria (CRGNB). The ability to simultaneously measure fosfomycin and other antibiotic drug levels will support in vitro and clinical investigations to develop rational antibiotic combination dosing regimens against CRGNB infections. We developed an analytical assay to measure fosfomycin with nine important antibiotics in human plasma and cation-adjusted Mueller–Hinton II broth (CAMHB). We employed a liquid-chromatography tandem mass spectrometry method and validated the method based on accuracy, precision, matrix effect, limit-of-detection, limit-of-quantification, specificity, carryover, and short-term and long-term stability on U.S. Food & Drug Administration (FDA) guidelines. Assay feasibility was assessed in a pilot clinical study in four patients on antibiotic combination therapy. Simultaneous quantification of fosfomycin, levofloxacin, meropenem, doripenem, aztreonam, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, cefepime, and tigecycline in plasma and CAMHB were achieved within 4.5 min. Precision, accuracy, specificity, and carryover were within FDA guidelines. Fosfomycin combined with any of the nine antibiotics were stable in plasma and CAMHB up to 4 weeks at −80 °C. The assay identified and quantified the respective antibiotics administered in the four subjects. Our assay can be a valuable tool for in vitro and clinical applications