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1.  Racial Disparities in Trajectories of Dental Caries Experience* 
This study charted the trajectories of dental caries, including decayed teeth, missing teeth, and filled teeth among older Americans over a 5-year period. In particular, it focused on racial differences in the levels of and rates of change in dental caries experience.
Data came from the Piedmont Dental Study. The sample included 810 older Americans who were dentate at the baseline with up to 4 repeated observations between 1988 and 1994. Hierarchical linear models were employed in depicting intrapersonal and interpersonal differences in dental caries experience.
Different measures of caries outcomes exhibited distinct trajectories. On average, the number of decayed teeth decreased over time, whereas missing teeth increased. In contrast, the number of filled teeth remained stable during a 5-year period. Relative to their white counterparts, older black Americans had more decayed teeth and missing teeth but fewer filled teeth. Blacks and whites differed in the levels of dental caries but not in their rates of change except for missing teeth. Even when demographic and socioeconomic attributes were adjusted, racial variations in dental caries experience remained significant.
Though significantly correlated, various dental caries outcomes move along different paths over time. In view of the persistent racial disparities in dental caries trajectories, future interventions to minimize such variations among older Americans in the levels of and the rates of change in dental caries experience are clearly warranted.
PMCID: PMC4324468  PMID: 23465078
2.  Phosphorene nanoribbon as a promising candidate for thermoelectric applications 
Scientific Reports  2014;4:6452.
In this work, the electronic properties of phosphorene nanoribbons with different width and edge configurations are studied by using density functional theory. It is found that the armchair phosphorene nanoribbons are semiconducting while the zigzag nanoribbons are metallic. The band gaps of armchair nanoribbons decrease monotonically with increasing ribbon width. By passivating the edge phosphorus atoms with hydrogen, the zigzag series also become semiconducting, while the armchair series exhibit a larger band gap than their pristine counterpart. The electronic transport properties of these phosphorene nanoribbons are then investigated using Boltzmann theory and relaxation time approximation. We find that all the semiconducting nanoribbons exhibit very large values of Seebeck coefficient and can be further enhanced by hydrogen passivation at the edge. Taking pristine armchair nanoribbons and hydrogen-passivated zigzag naoribbons with width N = 7, 8, 9 as examples, we calculate the lattice thermal conductivity with the help of phonon Boltzmann transport equation and evaluate the width-dependent thermoelectric performance. Due to significantly enhanced Seebeck coefficient and decreased thermal conductivity, we find that at least one type of phosphorene nanoribbons can be optimized to exhibit very high figure of merit (ZT values) at room temperature, which suggests their appealing thermoelectric applications.
PMCID: PMC4171703  PMID: 25245326
3.  Expression pattern of tumour-associated antigens in hepatocellular carcinoma: association with immune infiltration and disease progression 
British Journal of Cancer  2013;109(4):1031-1039.
The distinct expression pattern of tumour-associated antigens (TAAs) might be a critical reason for the inefficacy of immunity-based treatments and heterogeneous postsurgical recovery in patients with solid tumours, including hepatocellular carcinoma (HCC). However, little is known about the clinical value of the coexpression patterns of multiple TAAs.
We determined the expression of multiple TAAs with identified immunogenicity (GPC3, AFP, SSX-2, NY-ESO-1, EpCAM, midkine) and the density of tumour-infiltrating immune cells by immunohistochemistry in a panel of 362 primary HCC patients. We evaluated the association between the TAAs, immune cell infiltration, clinicopathological parameters, and prognosis.
Patients who coexpressed more TAAs had better prognosis (P<0.00001, overall survival). The integrated pattern of TAA was associated with good differentiation and small tumour size, and with more CD57+ natural killer and CD20+ B-cell infiltration (P<0.05). Multivariate Cox proportional hazards analysis identified the TAA index as an independent prognostic indicator (hazard ratio 0.625; 95% confidence interval 0.467–0.837; P=0.002), and could further predict patient prognosis in collaboration with local immune infiltration.
Our results could provide new evidence for the improvement of prognostic molecular signatures in HCC, and a novel rationale for patient enrolment in future immunotherapeutic trials and/or clinical treatments.
PMCID: PMC3749565  PMID: 23868000
tumour-associated antigen; immune infiltration; prognosis; hepatocellular carcinoma
4.  Chicken domestication: an updated perspective based on mitochondrial genomes 
Heredity  2012;110(3):277-282.
Domestic chickens (Gallus gallus domesticus) fulfill various roles ranging from food and entertainment to religion and ornamentation. To survey its genetic diversity and trace the history of domestication, we investigated a total of 4938 mitochondrial DNA (mtDNA) fragments including 2843 previously published and 2095 de novo units from 2044 domestic chickens and 51 red junglefowl (Gallus gallus). To obtain the highest possible level of molecular resolution, 50 representative samples were further selected for total mtDNA genome sequencing. A fine-gained mtDNA phylogeny was investigated by defining haplogroups A–I and W–Z. Common haplogroups A–G were shared by domestic chickens and red junglefowl. Rare haplogroups H–I and W–Z were specific to domestic chickens and red junglefowl, respectively. We re-evaluated the global mtDNA profiles of chickens. The geographic distribution for each of major haplogroups was examined. Our results revealed new complexities of history in chicken domestication because in the phylogeny lineages from the red junglefowl were mingled with those of the domestic chickens. Several local domestication events in South Asia, Southwest China and Southeast Asia were identified. The assessment of chicken mtDNA data also facilitated our understanding about the Austronesian settlement in the Pacific.
PMCID: PMC3668654  PMID: 23211792
chicken; mtDNA; domestication; phylogeny; Austronesian
5.  Quantum phases in circuit QED with a superconducting qubit array 
Scientific Reports  2014;4:4083.
Circuit QED on a chip has become a powerful platform for simulating complex many-body physics. In this report, we realize a Dicke-Ising model with an antiferromagnetic nearest-neighbor spin-spin interaction in circuit QED with a superconducting qubit array. We show that this system exhibits a competition between the collective spin-photon interaction and the antiferromagnetic nearest-neighbor spin-spin interaction, and then predict four quantum phases, including: a paramagnetic normal phase, an antiferromagnetic normal phase, a paramagnetic superradiant phase, and an antiferromagnetic superradiant phase. The antiferromagnetic normal phase and the antiferromagnetic superradiant phase are new phases in many-body quantum optics. In the antiferromagnetic superradiant phase, both the antiferromagnetic and superradiant orders can coexist, and thus the system possesses symmetry. Moreover, we find an unconventional photon signature in this phase. In future experiments, these predicted quantum phases could be distinguished by detecting both the mean-photon number and the magnetization.
PMCID: PMC3923215  PMID: 24522250
6.  Aphid Feeding Activates Expression of a Transcriptome of Oxylipin-Based Defense Signals in Wheat Involved in Resistance to Herbivory 
Journal of chemical ecology  2010;36(3):10.1007/s10886-010-9756-8.
Damage by the Russian wheat aphid (RWA), Diuraphis noxia, significantly reduces wheat and barley yields worldwide. In compatible interactions, virulent RWA populations flourish and susceptible plants suffer extensive leaf chlorophyll loss. In incompatible interactions, RWA reproduction and population growth are significantly reduced and RWA-related chlorophyll loss in resistant plants is minor. The objectives of this study were to develop an understanding of the molecular and phytochemical bases of RWA resistance in plants containing the Dnx resistance gene. Microarray, real-time polymerase chain reaction, and phytohormone assays were conducted to identify transcriptome components unique to RWA-infested Dnx plants and susceptible (Dn0) plants, and to identify and characterize putative genes involved in Dnx plant defense responses. We found that RWA-infested Dnx plants upregulated > 180 genes related to reactive oxygen species, signaling, pathogen defense, and arthropod allelochemical and physical defense. The expression of several of these genes in RWA-infested Dnx plants increased significantly from 6- to 24-h post infestation (hpi), but their expression in Dn0 plants, when present, was delayed until 48- to 96 hpi. Concentrations of 16- and 18-carbon fatty acids, trans-methyl-12-oxophytodienoic acid, and abscisic acid were significantly greater in Dnx foliage than in Dn0 foliage after RWA infestation, suggesting that Dnx RWA defense and resistance genes may be regulated via the oxylipin pathway. These findings provide a foundation for the elucidation of the molecular basis for compatible- and incompatible plant-aphid interactions.
PMCID: PMC3831272  PMID: 20229216
Diuraphis noxia; Insect; Microarray; Northern blot; Oxylipin signaling; Plant defense; Real-time polymerase chain reaction (PCR); Russian wheat aphid; Triticum aestivum; Wheat
7.  Orbit-induced spin squeezing in a spin-orbit coupled Bose-Einstein condensate 
Scientific Reports  2013;3:3166.
In recent pioneer experiment, a strong spin-orbit coupling, with equal Rashba and Dresselhaus strengths, has been created in a trapped Bose-Einstein condensate. Moreover, many exotic superfluid phenomena induced by this strong spin-orbit coupling have been predicted. In this report, we show that this novel spin-orbit coupling has important applications in quantum metrology, such as spin squeezing. We first demonstrate that an effective spin-spin interaction, which is the heart for producing spin squeezing, can be generated by controlling the orbital degree of freedom (i.e., the momentum) of the ultracold atoms. Compared with previous schemes, this realized spin-spin interaction has advantages of no dissipation, high tunability, and strong coupling. More importantly, a giant squeezing factor (lower than −30 dB) can be achieved by tuning a pair of Raman lasers in current experimental setup. Finally, we find numerically that the phase factor of the prepared initial state affects dramatically on spin squeezing.
PMCID: PMC3819609  PMID: 24196590
8.  Piperlongumine induces autophagy by targeting p38 signaling 
Cell Death & Disease  2013;4(10):e824-.
Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death.
PMCID: PMC3824668  PMID: 24091667
piperlongumine; autophagy; p38; reactive oxygen species
9.  Saccharomyces cerevisiae Live Cells Decreased In vitro Methane Production in Intestinal Content of Pigs 
An in vitro gas production technique was used in this study to elucidate the effect of two strains of active live yeast on methane (CH4) production in the large intestinal content of pigs to provide an insight to whether active live yeast could suppress CH4 production in the hindgut of pigs. Treatments used in this study include blank (no substrate and no live yeast cells), control (no live yeast cells) and yeast (YST) supplementation groups (supplemented with live yeast cells, YST1 or YST2). The yeast cultures contained 1.8×1010 cells per g, which were added at the rates of 0.2 mg and 0.4 mg per ml of the fermented inoculum. Large intestinal contents were collected from 2 Duroc×Landrace×Yorkshire pigs, mixed with a phosphate buffer (1:2), and incubated anaerobically at 39°C for 24 h using 500 mg substrate (dry matter (DM) basis). Total gas and CH4 production decreased (p<0.05) with supplementation of yeast. The methane production reduction potential (MRP) was calculated by assuming net methane concentration for the control as 100%. The MRP of yeast 2 was more than 25%. Compared with the control group, in vitro DM digestibility (IVDMD) and total volatile fatty acids (VFA) concentration increased (p<0.05) in 0.4 mg/ml YST1 and 0.2 mg/ml YST2 supplementation groups. Proportion of propionate, butyrate and valerate increased (p<0.05), but that of acetate decreased (p<0.05), which led to a decreased (p<0.05) acetate: propionate (A: P) ratio in the both YST2 treatments and the 0.4 mg/ml YST 1 supplementation groups. Hydrogen recovery decreased (p<0.05) with yeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (p<0.05) with yeast supplementation after 24 h of incubation. Our results suggest that live yeast cells suppressed in vitro CH4 production when inoculated into the large intestinal contents of pigs and shifted the fermentation pattern to favor propionate production together with an increased population of acetogenic bacteria, both of which serve as a competitive pathway for the available H2 resulting in the reduction of methanogenic archaea.
PMCID: PMC4093248  PMID: 25049860
In vitro Gas Production; Methane; Methanogenic Archaea; Pig; Saccharomyces cerevisiae; Volatile Fatty Acid
10.  Effects of Enzyme Treated Palm Kernel Expeller on Metabolizable Energy, Growth Performance, Villus Height and Digesta Viscosity in Broiler Chickens 
This study examined whether pre-treating palm kernel expeller (PKE) with exogenous enzyme would degrade its fiber content; thus improving its metabolizable energy (ME), growth performance, villus height and digesta viscosity in broiler chickens fed diets containing PKE. Our results showed that enzyme treatment decreased (p<0.05) hemicellulose and cellulose contents of PKE by 26.26 and 32.62%, respectively; and improved true ME (TME) and its nitrogen corrected value (TMEn) by 38% and 33%, respectively, compared to the raw sample. Average daily gain (ADG), feed intake and feed conversion ratio (FCR) of chickens fed on different dietary treatments in the grower period were not significantly different. Although there was no difference in feed intake (p>0.05) among treatment groups in the finisher period, ADG of chickens in the control (PKE-free diet) was higher (p<0.05) than in all treatment groups fed either 20 or 30% PKE, irrespective of with or without enzyme treatment. However, ADG of birds fed with 20% PKE was higher than those fed with 30% PKE. The FCR of chickens in the control was the lowest (2.20) but not significantly different from those fed 20% PKE diets while birds in the 30% PKE diets recorded higher (p>0.05) FCR. The intestinal villus height and crypt depth (duodenum, jejunum and ileum) were not different (p>0.05) among treatments except for duodenal crypt depth. The villus height and crypt depth of birds in enzyme treated PKE diets were higher (p<0.05) than those in the raw PKE groups. Viscosity of the intestinal digesta was not different (p>0.05) among treatments. Results of this study suggest that exogenous enzyme is effective in hydrolyzing the fiber (hemicellulose and cellulose) component and improved the ME values of PKE, however, the above positive effects were not reflected in the growth performance in broiler chickens fed the enzyme treated PKE compared to those received raw PKE. The results suggest that PKE can be included up to 5% in the grower diet and 20% in the finisher diet without any significant negative effect on FCR in broiler chickens.
PMCID: PMC4093377  PMID: 25049820
Palm Kernel Expeller; True Metabolizable Energy; Enzyme; Broiler
11.  Clinical Proof-of-Concept Study With MSDC-0160, a Prototype mTOT-Modulating Insulin Sensitizer 
It may be possible to achieve insulin sensitivity through the recently identified mitochondrial target of thiazolidinediones (mTOT), thereby avoiding peroxisome proliferator–activated receptor-γ (PPAR-γ)-dependent side effects. In this phase IIb clinical trial, 258 patients with type 2 diabetes completed a 12-week protocol with 50, 100, or 150 mg of MSDC-0160 (an mTOT modulator), 45 mg pioglitazone HCl (a PPAR-γ agonist), or a placebo. The two active treatments lowered fasting glucose levels to the same extent. The decreases in glycated hemoglobin (HbA1c) observed with the two higher doses of MSDC-0160 were not different from those associated with pioglitazone. By contrast, fluid retention as evidenced by reduction in hematocrit, red blood cells, and total hemoglobin was 50% less in the MSDC-0160–treated groups. There was also a smaller increase in high-molecular-weight (HMW) adiponectin with MSDC-0160 than with pioglitazone (P < 0.0001), suggesting that MSDC-0160 produces less expansion of white adipose tissue. Thus, mTOT modulators may have glucose-lowering effects similar to those of pioglitazone but without the adverse effects associated with PPAR-γ agonists.
PMCID: PMC3604641  PMID: 23462886
12.  Clinical trial: low- and standard-dose peginterferon alfa-2a for chronic hepatitis C, genotype 2 or 3 - efficacy, tolerability, viral kinetics and cytokine response 
Chronic infection with hepatitis C, genotype 2/3, responds better than other genotypes to peginterferon and ribavirin treatment. We hypothesized that a lower dose of peginterferon would be as effective, but less toxic than standard doses.
30 patients were treated with low-dose peginterferon alfa-2a (90 μg/week) and 27 patients with standard doses (180 μg/week) for 24 weeks in combination with 800 mg/day of ribavirin. Patients who failed treatment were offered 48 weeks of standard-dose treatment. Viral and serum IP-10 levels were measured and early viral kinetic parameters were calculated.
Sustained virological response was achieved in 68% of the low-dose and 87% of the standard-dose patients (per-protocol, p=0.79 for non-inferiority). Retreatment was successful in all patients who tolerated full dose and duration. The standard-dose group had greater first phase declines of viral levels and faster time to negativity. The second phase slope was not dose-dependent. IP-10 induction was significantly greater with the standard dose. Although fatigue and general feeling during treatment were worse for standard dose, hematologic toxicity and depression did not differ between groups.
A lower dose of peginterferon is associated with some symptomatic benefit but the response is not equivalent to standard dosing.
PMCID: PMC2861161  PMID: 20163377
Liver; Viral hepatitis
13.  Clinical trial: pilot study of metformin for the treatment of non-alcoholic steatohepatitis 
Non-alcoholic steatohepatitis (NASH) is a form of progressive fatty liver disease that is strongly associated with insulin resistance, which suggests that insulin sensitizing agents such as metformin may be beneficial for NASH.
To assess the effects of metformin on insulin sensitivity, body composition, serum alanine aminotransferase (ALT) levels and liver histology in patients with NASH.
Patients underwent liver biopsy, metabolic profiling and imaging studies before and at the end 48 weeks of metformin (2000 mg/day) therapy. The primary endpoint was a three-point improvement in the histological NASH activity index.
Of 28 patients enrolled, 26 (13 females; average age 44 years) completed 48 weeks of treatment and underwent repeat metabolic studies, imaging and liver biopsy. Thirty per cent achieved a histological response. Most patients lost weight, the average being 6 kg. There was a marked association between weight loss and improvements in NASH activity index and ALT levels (both, P < 0.01). Insulin sensitivity also improved, but the degree of change did not correlate with histological improvement.
Metformin leads to improvements in liver histology and ALT levels in 30% of patients with NASH, probably by its effects in causing weight loss.
PMCID: PMC2990687  PMID: 18945255
14.  Sustained elevation of Epstein–Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma 
British Journal of Cancer  2007;96(4):623-630.
We have monitored Epstein–Barr virus (EBV) IgA antibody levels of 39 nasopharyngeal carcinoma (NPC) cases for up to 15 years before clinical onset of NPC, and assessed preclinical serologic status of another 68 cases. Our results identify a serologic window preceding diagnosis when antibody levels are raised and sustained. This window can persist for as long as 10 years, with a mean duration estimated to as 37±28 months. Ninety-seven of these 107 NPC cases exhibited such a window. Cases that did not may reflect individual antibody response to EBV. Serologic screening at enrollment identified those cases who had already entered the window and became clinically manifested earlier (median=28 months) than those who entered the window after enrollment (median=90 months). The former account for 19 of 21 cases diagnosed within 2 years of screening. Nasopharyngeal carcinoma risk levels among seropositive subjects were also highest during this period. Both prediction rates and risk levels declined thereafter; cases detected at later times were composed of increasing proportions of individuals who entered the serological window after screening. Our findings establish EBV antibody as an early marker of NPC and suggest that repeated screening to monitor cases as they enter this window has considerable predictive value, with practical consequences for cancer treatment.
PMCID: PMC2360049  PMID: 17285127
NPC; EBV; serology; cancer screening; tumour marker
15.  Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. 
Journal of Clinical Investigation  1996;98(10):2268-2276.
Viral mutations have been implicated in alteration of the biological phenotype of hepatitis B virus (HBV). We recently cloned and sequenced the viral genome of an HBV strain associated with an outbreak of fulminant hepatitis (FH strain). The FH strain contained numerous mutations in all genomic regions and was functionally characterized by a more efficient encapsidation of pregenomic RNA leading to highly enhanced replication. To define the responsible mutation(s) for the enhanced replication, we introduced individual mutations of the FH strain into a wild-type construct by oligonucleotide-directed mutagenesis. Analysis of viral replication showed that two adjacent mutations in the HBV core promotor (C to T at nucleotide 1768 and T to A at nucleotide 1770) led to high level replication. Similar to the FH strain, this mutant displayed the phenotype of enhanced encapsidation of pregenomic RNA. Functional studies in an encapsidation assay demonstrated that the identified mutations resulted in a minor increase of pregenomic RNA transcription (two- to threefold) and a major transcription-independent enhancement (> 10-fold) of viral encapsidation. Our results demonstrate that the two adjacent mutations in the HBV core promotor region are responsible for the enhanced replication of the FH strain. These two mutations, outside the previously described encapsidation signal, core, and polymerase polypeptides, appeared to affect a novel genetic element involved in viral encapsidation.
PMCID: PMC507676  PMID: 8941643
16.  Proteasome complex as a potential cellular target of hepatitis B virus X protein. 
Journal of Virology  1996;70(8):5582-5591.
Although the biological importance of hepatitis B virus X protein (HBX) in the life cycle of hepatitis B virus has been well established, the cellular and molecular basis of its function remains largely undefined. Despite the association of multiple activities with HBX, none of them appear to provide a unifying hypothesis regarding the true biological function of HBX. Identification and characterization of cellular targets of HBX remain an essential goal in the elucidation of the molecular mechanisms of HBX. Using the Saccharomyces cerevisiae two-hybrid system, we have identified and characterized a novel subunit of the proteasome complex (XAPC7) that interacts specifically with HBX. We also showed that HBX binds specifically to XAPC7 in vitro. Mutagenesis studies have defined the domains of interaction to be critical for the function of HBX. Furthermore, overexpression of XAPC7 appeared to activate transcription by itself and antisense expression of XAPC7 was able to block transactivation by HBX. Therefore, the proteasome complex is possibly a functional target of HBX in cells.
PMCID: PMC190518  PMID: 8764072
17.  Transgenic expression of tpr-met oncogene leads to development of mammary hyperplasia and tumors. 
Journal of Clinical Investigation  1996;97(12):2872-2877.
Receptor tyrosine kinases are important in cell signal transduction and proliferation. Abnormal expression of tyrosine kinases often leads to malignant transformation. C-met is a tyrosine kinase receptor and its ligand is hepatocyte growth factor (HGF). HGF/c-met plays diverse role in regulation of cell growth, shape and movement. Constitutively activated met, such as tpr-met, is a potent oncogene in vitro, but its carcinogenic role in vivo remains unclear. Our study demonstrates that expression of tpr-met leads to development of mammary tumors and other malignancies in transgenic mice, and suggests that deregulated met expression may be involved in mammary carcinogenesis.
PMCID: PMC507382  PMID: 8675700
18.  Quantitative analysis of peptides from myelin basic protein binding to the MHC class II protein, I-Au, which confers susceptibility to experimental allergic encephalomyelitis. 
Molecular Medicine  1996;2(2):181-188.
BACKGROUND: An important issue in autoimmune diseases mediated by T cells, such as experimental allergic encephalomyelitis (EAE), is the affinity of the disease-inducing determinants for MHC class II proteins. Tolerance, either due to clonal deletion or anergy induction, is thought to require high-affinity interactions between peptides and MHC molecules. Low-affinity binding is compatible with the hypothesis that breaking tolerance to self proteins does not have to occur for onset of disease. In contrast, a high-affinity interaction implies that an event leading to a breakdown of tolerance is central to the autoimmune process. MATERIALS AND METHODS: Detergent-solubilized and affinity-purified I-Au was incubated with varying concentrations of a set of peptides from myelin basic protein and a biotinylated peptide agonist. The specific complexes were separated from excess peptide by capture on antibody-coated plates, and the affinity of the peptides was measured by adding europium-labeled streptavidin and measuring the resultant fluorescence. RESULTS: The immunodominant and encephalitogenic determinant, Ac 1-11, was shown to bind to I-Au relatively poorly (IC50 = 100 microM), demonstrating that in this protein, immunodominance did not correlate with high-affinity binding. In contrast with the natural sequence, the ability of shorter analogs to induce EAE did correlate with their apparent affinity. CONCLUSIONS: The dominance of the natural determinant does not arise from a high-affinity interaction with the MHC class II molecule. This suggests that other mechanisms are operative and that the specific T cell for this peptide/MHC ligand is of high affinity.
PMCID: PMC2230124  PMID: 8726461
19.  Endoscopic Ultrasonography Assessment for Ampullary and Bile Duct Malignancy 
From 1989 through 1992, endoscopic ultrasonography (EUS) was undertaken preoperatively to evaluate the extent of primary tumor, involvement of regional lymph nodes, and distant metastases in 22 patients with ampullary carcinoma and 18 patients with bile duct carcinoma. The results were compared with histopathological findings according to the TNM staging system. The accurate rate in assessing the extent of cancer invasion was 82% for ampullary carcinoma, 66% for common hepatic duct carcinoma, and 78% for common bile duct carcinoma. The accuracy of EUS in predicting regional lymph node metastasis was 59% for ampullary carcinoma, 56% for common hepatic duct carcinoma, and 67% for common bile duct carcinoma. Invasion of the portal vein was correctly predicted by EUS in 2 of 3 patients. None of the 3 patients with liver metastasis was detected by EUS. Therefore, endoscopic ultrasonography is an effective method in the evaluation of the extent of cancer invasion of ampullary and bile duct carcinoma as well as the involvement of regional lymph nodes preoperatively. However, due to its limited penetration depth, EUS is inadequate in the assessment of liver metastasis.
PMCID: PMC2362552  PMID: 18493415
20.  Enhanced replication of a hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. 
Journal of Virology  1994;68(3):1651-1659.
Hepatitis B virus (HBV) mutants unable to synthesize HBV e antigen have been described in association with fulminant hepatitis. We have cloned and sequenced the entire viral genome of an HBV strain associated with an epidemic of fulminant hepatitis. This strain contained, in addition to two G-to-A mutations in the precore region (nucleotides 1898 and 1901), numerous other mutations in conserved nucleotide positions resulting in significant amino acid substitutions in HBV gene products. We introduced either or both of the two G-to-A mutations into wild-type HBV by oligonucleotide-directed mutagenesis and generated replication-competent constructs of these mutants as well as the fulminant strain. Viral antigen synthesis, transcription, and replication were analyzed after transfection into human hepatoma cells. All viral constructs produced and secreted similar levels of envelope proteins (HBV surface antigen). Analysis of cellular lysate for core-specific immunoreactivity demonstrated a much higher level of core-associated antigens in cells transfected with the fulminant strain. While cells transfected with mutant and wild-type HBV DNAs synthesized similar levels of viral RNAs, the fulminant strain directed the synthesis of a much higher level of core-associated replicative intermediates (as well as virion particles) than the wild type and mutants with either or both of the precore mutations. Increase in the encapsidation of pregenomic RNA into core particles likely the basis for the enhanced replication associated with the fulminant strain. Our study suggests that an HBV mutant with enhanced viral replication may be important in the pathogenesis of fulminant hepatic failure, and mutations other than the precore mutations may be responsible for this variant behavior.
PMCID: PMC236623  PMID: 8107226
21.  Hepatitis B virus precore mutation and fulminant hepatitis in the United States. A polymerase chain reaction-based assay for the detection of specific mutation. 
Journal of Clinical Investigation  1994;93(2):550-555.
Hepatitis B virus (HBV) variants with precore mutation(s) resulting in the absence of HBeAg production have been associated with the occurrence of fulminant hepatitis in Japan, Israel, and southern Europe, where the prevalence of this HBV strain appears common. In areas such as United States, where HBV infection is not endemic, the role of this mutant virus in fulminant hepatitis is unknown. We developed an amplification refractory mutation detection system to detect specifically the presence of the G to A mutation at nucleotide position 1898, which is the most frequently observed mutation resulting in a precore stop codon. In addition, this method provided a quantitative measurement of the relative ratio of one strain to the other. Using this system, we tested HBV strains for the presence of the stop codon mutation in sera from 40 cases of fulminant hepatitis B occurring in the United States. Serum HBV DNAs from 28 patients were analyzed successfully. A mixture of wild-type and mutant strains in various ratios were observed in 15 patients, wild type exclusively in 11, and mutant exclusively in 2. Four of these patients had undergone liver transplantation for HBV-associated cirrhosis and developed fulminant HBV-associated hepatitis after transplantation. Pre- and posttransplant serum samples from one patient were analyzed: a mixture of wild-type and mutant HBV strains was detected in both samples. Our study demonstrated that both wild-type and mutant HBV strains are associated with fulminant hepatitis, and that in some patients in the United States, factors other than precore mutations contribute to the development of fulminant hepatitis.
PMCID: PMC293877  PMID: 8113393
22.  A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products. 
Molecular and Cellular Biology  1993;13(12):7476-7486.
Many viruses possess complex mechanisms involving multiple gene products and cis-regulatory elements in order to achieve a fine control of their gene expression at both transcriptional and posttranscriptional levels. Hepatitis B virus (HBV) and retroviruses share many structural and functional similarities. In this study, by genetic and biochemical analyses, we have demonstrated the existence of a novel genetic element within the HBV genome which is essential for high-level expression of viral gene products. This element is located 3' to the envelope coding region. We have shown that this genetic element is cis acting at the posttranscriptional level and that its function is exerted at the level of RNA processing as part of transcribed sequences. This RNA element is also functional in the context of a heterologous gene. Similar to the function of Rev-Rev response element interaction of human immunodeficiency virus type 1, this element appears to inhibit the splicing process and facilitate the transport and utilization of HBV transcripts.
PMCID: PMC364819  PMID: 8246965
23.  The patB gene product, required for growth of the cyanobacterium Anabaena sp. strain PCC 7120 under nitrogen-limiting conditions, contains ferredoxin and helix-turn-helix domains. 
Journal of Bacteriology  1993;175(6):1697-1704.
A mutant of Anabaena sp. strain PCC 7120, called PAT-2, that grows poorly under nitrogen-fixing conditions, has been isolated. The heterocysts of the mutant strain develop much more slowly than those of the wild type and are spaced more closely in an older culture of the mutant than in the wild type. The wild-type gene that complements the mutation in PAT-2, called patB, was isolated and characterized. The predicted 529-amino-acid PatB protein contains a region very similar to the Fe4S4 bacterial-type ferredoxins near its N terminus and a helix-turn-helix motif near its C terminus. This pattern of domains resembles those of transcriptional regulators in both prokaryotes and eukaryotes. The mutation in strain PAT-2 is the deletion of G at nucleotide 1342 in the patB gene, resulting in the loss of a 62-amino-acid fragment from the C terminus of the PatB protein, including the helix-turn-helix motif.
PMCID: PMC203964  PMID: 8449877
24.  Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells. 
Journal of Clinical Investigation  1993;91(3):1241-1246.
A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions.
PMCID: PMC288084  PMID: 8383700
25.  Hepatitis B virus X protein is not central to the viral life cycle in vitro. 
Journal of Virology  1992;66(2):1223-1227.
The hepatitis B x (HBx) gene is the smallest open reading frame of the hepatitis B virus (HBV) genome. It is conserved among all mammalian hepadnaviruses and is expressed during viral infection. While the HBx protein (pX) has been shown to trans-activate the transcription of a wide range of viral and cellular genes and to induce liver cancer in transgenic mice, the significance of pX for the life cycle of HBV itself has not been elucidated. To assess the function of pX in viral replication and virion export, we designed an X-minus mutant by introduction of a stop codon at the beginning of the HBx gene without affecting the viral polymerase gene product. Transient transfection analyses using different cell lines revealed that this X-minus mutant directs the synthesis of wild-type levels of viral proteins, replicative intermediates, and virion export. These data suggest that the expression of the highly conserved HBx gene is not central for the life cycle of HBV in vitro but may be involved in the pathogenicity of hepadnavirus infection, including liver cancer development.
PMCID: PMC240832  PMID: 1731101

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