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1.  Dual vulnerability of TDP-43 to calpain and caspase-3 proteolysis after neurotoxic conditions and traumatic brain injury 
Transactivation response DNA-binding protein 43 (TDP-43) proteinopathy has recently been reported in chronic traumatic encephalopathy, a neurodegenerative condition linked to prior history of traumatic brain injury (TBI). While TDP-43 appears to be vulnerable to proteolytic modifications under neurodegenerative conditions, the mechanism underlying the contribution of TDP-43 to the pathogenesis of TBI remains unknown. In this study, we first mapped out the calpain or caspase-3 TDP-43 fragmentation patterns by in vitro protease digestion. Concurrently, in cultured cerebrocortical neurons subjected to cell death challenges, we identified distinct TDP-43 breakdown products (BDPs) of 35, 33, and 12 kDa that were indicative of dual calpain/caspase attack. Cerebrocortical culture incubated with calpain and caspase-fragmented TDP-43 resulted in neuronal injury. Furthermore, increased TDP-43 BDPs as well as redistributed TDP-43 from the nucleus to the cytoplasm were observed in the mouse cortex in two TBI models: controlled cortical impact injury and overpressure blast-wave-induced brain injury. Finally, TDP-43 and its 35 kDa fragment levels were also elevated in the cerebrospinal fluid (CSF) of severe TBI patients. This is the first evidence that TDP-43 might be involved in acute neuroinjury and TBI pathology, and that TDP-43 and its fragments may have biomarker utilities in TBI patients.
PMCID: PMC4158661  PMID: 24917042
calpain; caspase; neurodegeneration; TDP-43; traumatic brain injury
2.  Interferons: Success in anti-viral immunotherapy 
Cytokine & growth factor reviews  2014;25(4):369-376.
The interferons (IFNs) are glycoproteins with strong antiviral activities that represent one of the first lines of host defense against invading pathogens. These proteins are classified into three groups, Type I, II and III IFNs, based on the structure of their receptors on the cell surface. Due to their ability to modulate immune responses, they have become attractive therapeutic options to control chronic virus infections. In combination with other drugs, Type I IFNs are considered a “standard of care” in suppressing Hepatitis C (HCV) and Hepatitis B (HBV) infections, while Type III IFN has generated encouraging results as a treatment for HCV infection in phase III clinical trials. However, though effective, using IFNs as a treatment is not without the need for caution. IFNs are such powerful cytokines that affect a wide array of cell types; as a result, patients usually experience unpleasant symptoms, with a percentage of patients suffering system wide effects. Thus, constant monitoring is required for patients treated with IFN in order to reach the treatment goals of suppressing virus infection and maintaining quality of life.
PMCID: PMC4182113  PMID: 25156421
Antiviral therapy; IFN-α/β; IFN-γ; IFN-λ
3.  Lactoferrin Induces Osteoblast Growth through IGF-1R 
Objectives. To investigate the role of the IGF-1R by which lactoferrin induces osteoblast growth. Methods. Osteoblast received 5 d lactoferrin intervention at a concentration of 0.1, 1, 10, 100, and 1000 μg/mL, and the IGF-1 and IGF-1R were detected using RT-PCR and western blot. The osteoblast into the control, 100 μg/mL lactoferrin, Neo-scramble (NS, empty vector), NS + 100 μg/mL lactoferrin, shIGF-1R and shIGF-1R + 100 μg/mL lactoferrin group. We test the apoptosis and proliferation and the level of PI3K and RAS in osteoblasts after 5 d intervention. Results. (1) 1, 10, 100, and 1000 μg/mL lactoferrin induced the expression of IGF-1 mRNA and protein. 10 μg/mL and 100 μg/mL lactoferrin induced the expression of IGF-1R mRNA and protein. (2) Lactoferrin (100 μg/mL) induced osteoblast proliferation while inhibiting apoptosis. Osteoblasts with silenced IGF-1R exhibited decreased proliferation but increased apoptosis. MMT staining and flow cytometry both indicated that there was no significant difference between the shIGF-1R group and the shIGF-1R + 100 μg/mL lactoferrin group. (3) Lactoferrin (100 μg/mL) induced PI3K and RAS phosphorylation and silence of IGF-1R resulted in decreased p-PI3K and p-RAS expression. Lactoferrin-treated shIGF-1R cells showed significantly higher level of p-PI3K and p-RAS when compared with shIGF-1R. Conclusion. Lactoferrin induced IGF-1/IGF-1R in a concentration-dependent manner. Lactoferrin promoted osteoblast proliferation while inhibiting apoptosis through IGF-1R. Lactoferrin activated PI3K and RAS phosphorylation via an IGF-1R independent pathway.
PMCID: PMC4531176  PMID: 26290662
4.  Temporal MRI characterization, neurobiochemical and neurobehavioral changes in a mouse repetitive concussive head injury model 
Scientific Reports  2015;5:11178.
Single and repeated sports-related mild traumatic brain injury (mTBI), also referred to as concussion, can result in chronic post-concussive syndrome (PCS), neuropsychological and cognitive deficits, or chronic traumatic encephalopathy (CTE). However PCS is often difficult to diagnose using routine clinical, neuroimaging or laboratory evaluations, while CTE currently only can be definitively diagnosed postmortem. We sought to develop an animal model to simulate human repetitive concussive head injury for systematic study. In this study, mice received single or multiple head impacts by a stereotaxic impact device with a custom-made rubber tip-fitted impactor. Dynamic changes in MRI, neurobiochemical markers (Tau hyperphosphorylation and glia activation in brain tissues) and neurobehavioral functions such as anxiety, depression, motor function and cognitive function at various acute/subacute (1-7 day post-injury) and chronic (14-60 days post-injury) time points were examined. To explore the potential biomarkers of rCHI, serum levels of total Tau (T-Tau) and phosphorylated Tau (P-Tau) were also monitored at various time points. Our results show temporal dynamics of MRI consistent with structural perturbation in the acute phase and neurobiochemical changes (P-Tau and GFAP induction) in the subacute and chronic phase as well as development of chronic neurobehavioral changes, which resemble those observed in mTBI patients.
PMCID: PMC4461921  PMID: 26058556
5.  Gene Transfer and Genome-Wide Insertional Mutagenesis by Retroviral Transduction in Fish Stem Cells 
PLoS ONE  2015;10(6):e0127961.
Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.
PMCID: PMC4451014  PMID: 26029933
6.  A microrna screen to identify regulators of peritoneal fibrosis in a rat model of peritoneal dialysis 
BMC Nephrology  2015;16:48.
Peritoneal fibrosis is a common complication in patients treated with long-term peritoneal dialysis. The aim of this study was to identify the microRNAs (miRNAs) involved in regulation of peritoneal fibrosis in a rat model of peritoneal dialysis.
Twenty-four Sprague–Dawley (SD) rats were randomly allocated into three groups: (i) Control group (Cg, n = 8); (ii) Saline group (Sg, n = 8): daily intraperitoneal injection with 0.9% normal saline; (iii) Hypertonic dialysate group (HDg, n = 8): daily intraperitoneal injection with 4.25% peritoneal dialysis solution. Rats were sacrificed after four weeks for histological evaluation of peritoneal membrane and the expression of α-SMA and COL-1. A miRNA screen was performed using microarray analysis to identify differentially expressed miRNAs, which were then validated by real-time PCR.
Compared with the control and the saline groups, hypertonic dialysate group showed impaired peritoneal function accompanied by a spectrum of morphological changes including thicker peritoneal membrane, higher collagen deposition, infiltration of mononuclear cells and neovascularization in the peritoneum. Increased mRNA and protein levels of α-SMA and COL-1 were observed in hypertonic dialysate group, indicating the progression of peritoneal fibrosis. The miRNA screen identified 8 significantly down-regulated miRNAs (miR-31, miR-93, miR-100, miR-152, miR-497, miR-192, miR-194 and miR-200b) and one highly up-regulated miRNA (miR-122) in the hypertonic dialysate group. The results were confirmed by real-time PCR.
Altered miRNA expression in peritoneum was found in the rat model of peritoneal fibrosis, indicating that these miRNAs may be associated with pathogenesis of peritoneal fibrosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12882-015-0039-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4546227  PMID: 25884636
Peritoneal fibrosis; miRNA; Peritoneal dialysis; Epithelial-to-mesenchymal transition
7.  The Relationship of Retinal Vessel Diameters and Fractal Dimensions with Blood Pressure and Cardiovascular Risk Factors 
PLoS ONE  2014;9(9):e106551.
This study aimed to investigate the correlation between quantitative retinal vascular parameters such as central retinal arteriolar equivalent (CRAE) and retinal vascular fractal dimension (D(f)), and cardiovascular risk factors in the Chinese Han population residing in the in islands of southeast China.
Methodology/Principle Findings
In this cross-sectional study, fundus photographs were collected and semi-automated analysis software was used to analyze retinal vessel diameters and fractal dimensions. Cardiovascular risk factors such as relevant medical history, blood pressure (BP), lipids, and blood glucose data were collected. Subjects had a mean age of 51.9±12.0 years and included 812 (37.4%) males and 1,357 (62.6%) females. Of the subjects, 726 (33.5%) were overweight, 226 (10.4%) were obese, 272 (12.5%) had diabetes, 738 (34.0%) had hypertension, and 1,156 (53.3%) had metabolic syndrome. After controlling for the effects of potential confounders, multivariate analyses found that age (β = 0.06, P = 0.008), sex (β = 1.33, P = 0.015), mean arterial blood pressure (β = −0.12, P<0.001), high-sensitivity C-reactive protein (β = −0.22, P = 0.008), and CRVE (β = 0.23, P<0.001) were significantly associated with CRAE. Age (β = −0.0012, P<0.001), BP classification (prehypertension: β = −0.0075, P = 0.014; hypertension: β = −0.0131, P = 0.002), and hypertension history (β = −0.0007, P = 0.009) were significantly associated with D(f).
D(f) exhibits a stronger association with BP than CRAE. Thus, D(f) may become a useful indicator of cardiovascular risk.
PMCID: PMC4154709  PMID: 25188273
8.  Brain Susceptibility Weighted Imaging Signal Changes in Acute Hemorrhagic Anemia: An Experimental Study Using a Rabbit Model 
The aim of this study was to investigate susceptibility-weighted imaging (SWI) signal changes in different brain regions in a rabbit model of acute hemorrhagic anemia.
Ten New Zealand white rabbits were used for construction of the model of acute hemorrhagic anemia. Signal intensities of SWI images of the bilateral frontal cortex, frontal white matter, temporal lobe, and thalamic nuclei were measured. In addition, the cerebral gray-white contrast and venous structures of the SWI images were evaluated by an experienced physician.
Repeated bloodletting was associated with significant reductions in red blood cell count, hemoglobin concentration, hematocrit, pH, and PaCO2, and elevations of blood lactate and PaO2. In normal status, the SWI signal intensity was significantly higher in the frontal cortex than in the frontal white matter (63.10±22.82 vs. 52.50±20.29; P<0.05). Repeated bloodletting (5 occasions) caused significant (P<0.05) decreases in the SWI signals of the frontal cortex (from 63.10±22.82 to 37.70±4.32), temporal lobe (from 52.50±20.29 to 42.60±5.54), and thalamus (from 60.40±20.29 to 39.40±3.47), but was without effect in the frontal white matter. The cerebral white-gray contrast and venous structures were clearer after bloodletting than before bloodletting.
The effect of hemorrhage on the brain is reflected by SWI signal changes in the cerebral cortex and gray matter nuclei.
PMCID: PMC4116343  PMID: 25060330
Anemia; Cholangiopancreatography; Magnetic Resonance; Hemoglobin, Sickle
9.  Improved endoscopic retrograde cholangiopancreatography brush increases diagnostic yield of malignant biliary strictures 
AIM: To determine if a new brush design could improve the diagnostic yield of biliary stricture brushings.
METHODS: Retrospective chart review was performed of all endoscopic retrograde cholangiopancreatography procedures with malignant biliary stricture brushing between January 2008 and October 2012. A standard wire-guided cytology brush was used prior to protocol implementation in July 2011, after which, a new 9 French wire-guided cytology brush (Infinity sampling device, US Endoscopy, Mentor, OH) was used for all cases. All specimens were reviewed by blinded pathologists who determined whether the sample was positive or negative for malignancy. Cellular yield was quantified by describing the number of cell clusters seen.
RESULTS: Thirty-two new brush cases were compared to 46 historical controls. Twenty-five of 32 (78%) cases in the new brush group showed abnormal cellular findings consistent with malignancy as compared to 17 of 46 (37%) in the historical control group (P = 0.0003). There was also a significant increase in the average number of cell clusters of all sizes (21.1 vs 9.9 clusters, P = 0.0007) in the new brush group compared to historical controls.
CONCLUSION: The use of a new brush design for brush cytology of biliary strictures shows increased diagnostic accuracy, likely due to improved cellular yield, as evidenced by an increase in number of cellular clusters obtained.
PMCID: PMC4094989  PMID: 25031790
Malignant biliary stricture; Endoscopic retrograde cholangiopancreatography; Brush cytology; Diagnostic yield; Cytopathology
10.  A propensity score analysis on the effect of on-pump versus off-pump coronary artery bypass grafting for patients with coronary artery disease 
Journal of Thoracic Disease  2014;6(6):838-844.
The aim of this retrospective observational study was to investigate the effect of on-pump versus off-pump coronary artery bypass grafting (CABG) for patients with coronary artery diseases (CAD).
A retrospective observational study was performed using a propensity score analysis in 290 consecutive patients undergoing CABG between April 2009 and March 2014, of them, 54 patients undergoing off-pump CABG (OPCABG) were matched with 54 patients undergoing on-pump CABG (ONCABG) by propensity score. The perioperative complications and hospital mortality were documented.
Preoperative characteristics were comparable in both groups following propensity matching. Postoperative myocardial infarction (MI) incidence was lower in OPCABG group than in ONCABG group (3.7% vs. 14.8%, P=0.046); both hospital mortality and the major complications rates were similar in the two groups after propensity adjustment for preoperative characteristics.
The perioperative complications are similar in both off-pump and on pump CABG groups, the short-term effect of OPCABG is similar to that of ONCABG.
PMCID: PMC4073416  PMID: 24977010
Coronary artery disease (CAD); coronary artery bypass grafting (CABG); cardiopulmonary bypass (CPB); propensity score analysis
12.  Effect of thalidomide on the proliferation of hepatoma cells assessed by osteopontin levels in nude mice 
The aim of the present study was to investigate the inhibitory effects of thalidomide in the hepatocellular carcinoma nude mouse model in order to provide new insights into a comprehensive clinical intervention for hepatocellular carcinoma. MHCC97 cells were routinely cultured, passaged and adjusted to a single cell suspension with a concentration of 2×107/ml. Six-week-old, BALB/C male nude mice were anesthetized and fixed in the prone position, then a subcapsular injection of the single cell suspension was administered into the spleen and their abdomens were closed. A laparotomy and left hepatic lobectomy was performed 14 days later and the abdomens were closed once again. Subsequent to the establishment of the hepatocellular carcinoma model, the nude mice were randomly divided into three groups, each consisting of 12 mice. The early intervention group were immediately provided with the post-operative thalidomide intervention, the late intervention group were provided with the post-operative thalidomide intervention one week subsequent to the surgery, and the negative control group were provided with a placebo intervention (0.9% physiological saline). Each intervention was continuously administered once per day for one week. The osteopontin (OPN) content of the liver tumors was detected using immunohistochemistry. The data were analyzed using an analysis of variance (ANOVA) test. There were significant differences in the OPN levels of the tumors among the early intervention, late intervention and negative control groups. Thalidomide may inhibit the generation of OPN and thereby inhibit the infiltration and metastasis of tumors; the immediate use of thalidomide following hepatectomy in the present study may block the invasion and metasis for liver cancer more effectively.
PMCID: PMC3671833  PMID: 23737889
osteopontin; thalidomide; hepatocellular carcinoma
13.  Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis 
Nature Genetics  2012;44(5):562-569.
Variants associated with meconium ileus in cystic fibrosis (CF) were identified in 3,763 patients by GWAS. Five SNPs at two loci near SLC6A14 (min P=1.28×10−12 at rs3788766), chr Xq23-24 and SLC26A9 (min P=9.88×10−9 at rs4077468), chr 1q32.1 accounted for ~5% of the phenotypic variability, and were replicated in an independent patient collection (n=2,372; P=0.001 and 0.0001 respectively). By incorporating that disease-causing mutations in CFTR alter electrolyte and fluid flux across epithelia into an hypothesis-driven genome-wide analysis (GWAS-HD), we identified the same SLC6A14 and SLC26A9 associated SNPs, while establishing evidence for the involvement of SNPs in a third solute carrier gene, SLC9A3. In addition, GWAS-HD provided evidence of association between meconium ileus and multiple constituents of the apical plasma membrane where CFTR resides (P=0.0002, testing 155 apical genes jointly and replicated, P=0.022). These findings suggest that modulating activities of apical membrane constituents could complement current therapeutic paradigms for cystic fibrosis.
PMCID: PMC3371103  PMID: 22466613
14.  Transient and Stable GFP Expression in Germ Cells by the vasa Regulatory Sequences from the Red Seabream (Pagrus major) 
Primordial germ cells (PGCs) are the precursors of gametes responsible for genetic transmission to the next generation. They provide an ideal system for cryopreservation and restoration of biodiversity. Recently, considerable attention has been raised to visualize, isolate and transplant PGCs within and between species. In fish, stable PGC visualization in live embryo and individual has been limited to laboratory fish models such as medaka and zebrafish. One exception is the rainbow trout, which represents the only species with aquaculture importance and has GFP-labeled germ cells throughout development. PGCs can be transiently labeled by embryonic injection of mRNA containing green fluorescence protein gene (GFP) and 3'-untranslated region (3'-UTR) of a maternal germ gene such as vasa, nos1, etc. Stable PGC labeling can be achieved through production of transgenic animals by some transcriptional regulatory sequences from germ genes, such as the vasa promoter and 3'-UTR. In this study, we reported the functional analyses of the red seabream vasa (Pmvas) regulatory sequences, using medaka as a model system. It was showed that injection of GFP-Pmvas3'UTR mRNA was able to label medaka PGCs during embryogenesis. Besides, we have constructed pPmvasGFP transgenic vector, and established a stable transgenic medaka line exhibiting GFP expression in germ cells including PGCs, mitotic and meiotic germ cells of both sexes, under control of the Pmvas transcriptional regulatory sequences. It is concluded that the Pmvas regulatory sequences examined in this study are sufficient for germ cell expression and labeling.
PMCID: PMC3385010  PMID: 22745578
Pagrus major; PGCs; vasa; transgene; GFP
15.  FXYD3: A Promising Biomarker for Urothelial Carcinoma 
Biomarker Insights  2011;6:17-26.
Urothelial carcinoma (UC) of the kidney is a relatively rare but aggressive form of kidney cancer. Differential diagnosis of renal UC from renal cell carcinoma (RCC) can be difficult, but is critical for correct patient management. We aimed to use global gene expression profiling to identify genes specifically expressed in urothelial carcinoma (UC) of the kidney, with purpose of finding new biomarkers for differential diagnosis of UC of both upper and lower tract from normal tissues.
Materials and methods:
Microarray gene expression profiling was performed on a variety of human kidney tumor samples, including clear cell, papillary, chromophobe, oncocytoma, renal UC and normal kidney controls. Differentially expressed mRNAs in various kidney tumor subtypes were thus identified. Protein expression in human UC tumor samples from both upper and lower urinary tract was evaluated by immunohistochemistry.
FXYD3 (MAT-8) mRNA was specifically expressed in UC of the kidney and not in normal kidney tissue or in any RCC tumor subtypes. FXYD3 mRNA levels displayed equal or better prediction rate for the detection of renal UC than the mRNA levels of selected known UC markers as p63, vimentin, S100P, KRT20 and KRT7. Finally, immunohistochemical staining of clinical UC samples showed that FXYD3 protein is overexpressed in majority of UC of the upper genitourinary tract (encompassing the kidney, ∼90%) and in majority of high grade bladder UC (∼84%, it’s <40% in low grade tumors, P < 0.001) compared to normal kidney and bladder tissues.
FXYD3 may be a promising novel biomarker for the differential diagnosis of renal UC and a promising prognosis marker of UC from bladder. Because it was identified genome-widely, FXYD3 may have important biological ramifications for the genetic study of UC.
PMCID: PMC3076016  PMID: 21499437
FXYD3; FXYD; urothelial carcinoma; kidney; marker; microarray
16.  Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results 
Human genetics  2009;126(6):763-778.
Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.
PMCID: PMC2888886  PMID: 19662435
17.  Involvement of Histone Demethylase LSD1 in Blimp-1-Mediated Gene Repression during Plasma Cell Differentiation▿  
Molecular and Cellular Biology  2009;29(6):1421-1431.
Plasma cell differentiation is orchestrated by the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1), which silences the gene expression program of mature B cells. The molecular mechanism underlying Blimp-1 suppression of mature B-cell gene expression is not fully understood. Here we report that a proline-rich domain in Blimp-1 directly interacts with LSD1, a histone lysine demethylase. Both LSD1 knockdown and expression of Blimp-1 lacking the proline-rich domain derepressed the activities of Blimp-1-dependent luciferase reporters. Disruption of the Blimp-1 interaction with LSD1 or reduced LSD1 expression attenuated antibody production, demonstrating the biological significance of this interaction. Finally, using chromatin immunoprecipitation, we showed that Blimp-1 binding to its target sites is accompanied by LSD1 binding to those same sites and that LSD1 binding correlates with histone modifications of accessible chromatin. These findings provide further insights into the molecular mechanism of the silencing of mature B-cell genes by Blimp-1 in plasma cell differentiation.
PMCID: PMC2648243  PMID: 19124609
18.  CAPIH: A Web interface for comparative analyses and visualization of host-HIV protein-protein interactions 
BMC Microbiology  2009;9:164.
The Human Immunodeficiency Virus type one (HIV-1) is the major causing pathogen of the Acquired Immune Deficiency Syndrome (AIDS). A large number of HIV-1-related studies are based on three non-human model animals: chimpanzee, rhesus macaque, and mouse. However, the differences in host-HIV-1 interactions between human and these model organisms have remained unexplored.
Here we present CAPIH (Comparative Analysis of Protein Interactions for HIV-1), the first web-based interface to provide comparative information between human and the three model organisms in the context of host-HIV-1 protein interactions. CAPIH identifies genetic changes that occur in HIV-1-interacting host proteins. In a total of 1,370 orthologous protein sets, CAPIH identifies ~86,000 amino acid substitutions, ~21,000 insertions/deletions, and ~33,000 potential post-translational modifications that occur only in one of the four compared species. CAPIH also provides an interactive interface to display the host-HIV-1 protein interaction networks, the presence/absence of orthologous proteins in the model organisms in the networks, the genetic changes that occur in the protein nodes, and the functional domains and potential protein interaction hot sites that may be affected by the genetic changes. The CAPIH interface is freely accessible at
CAPIH exemplifies that large divergences exist in disease-associated proteins between human and the model animals. Since all of the newly developed medications must be tested in model animals before entering clinical trials, it is advisable that comparative analyses be performed to ensure proper translations of animal-based studies. In the case of AIDS, the host-HIV-1 protein interactions apparently have differed to a great extent among the compared species. An integrated protein network comparison among the four species will probably shed new lights on AIDS studies.
PMCID: PMC2782265  PMID: 19674441
19.  Lower Levels of Gamma Interferon Expressed by a Pseudotyped Single-Cycle Simian Immunodeficiency Virus Enhance Immunogenicity in Rats▿  
Journal of Virology  2008;83(4):1592-1601.
A vaccine for human immunodeficiency virus (HIV) infection is desperately needed to control the AIDS pandemic. To address this problem, we constructed single-cycle simian immunodeficiency viruses (SIVs) pseudotyped with the glycoprotein of vesicular stomatitis virus and expressing different levels of gamma interferon (IFN-γ) as a potential vaccine strategy. We previously showed that IFN-γ expression by pseudotyped SIVs does not alter viral single-cycle infectivity. T cells primed with dendritic cells transduced by pseudotyped SIVs expressing high levels of IFN-γ had stronger T-cell responses than those primed with dendritic cells transduced by constructs lacking IFN-γ. In the present study, we tested the immunogenicities of these pseudotyped SIVs in a rat model. The construct expressing low levels of rat IFN-γ (dSIVLRγ) induced higher levels of cell-mediated and humoral immune responses than the construct lacking IFN-γ (dSIVR). Rats vaccinated with dSIVLRγ also had lower viral loads than those vaccinated with dSIVR when inoculated with a recombinant vaccinia virus expressing SIV Gag-Pol as a surrogate challenge. The construct expressing high levels of IFN-γ (dSIVHRγ) did not further enhance immunity and was less protective than dSIVLRγ. In conclusion, the data indicated that IFN-γ functioned as an adjuvant to augment antigen-specific immune responses in a dose- and cell type-related manner in vivo. Thus, fine-tuning of the cytokine expression appears to be essential in designing vaccine vectors expressing adjuvant genes such as the gene for IFN-γ. Furthermore, we provide evidence of the utility of the rat model to evaluate the immunogenicities of single-cycle HIV/SIV recombinant vaccines before initiating studies with nonhuman primate models.
PMCID: PMC2643767  PMID: 19073726
20.  Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity▿  
Journal of Virology  2008;83(3):1216-1227.
A vaccine for the prevention of human immunodeficiency virus (HIV) infection is desperately needed to control the AIDS pandemic. To address this problem, we developed vesicular stomatitis virus glycoprotein-pseudotyped replication-defective simian immunodeficiency viruses (dSIVs) as an AIDS vaccine strategy. The dSIVs retain characteristics of a live attenuated virus without the drawbacks of potential virulence caused by replicating virus. To improve vaccine immunogenicity, we incorporated CD40 ligand (CD40L) into the dSIV envelope. CD40L is one of the most potent stimuli for dendritic cell (DC) maturation and activation. Binding of CD40L to its receptor upregulates expression of major histocompatibility complex class I, class II, and costimulatory molecules on DCs and increases production of proinflammatory cytokines and chemokines, especially interleukin 12 (IL-12). This cytokine polarizes CD4+ T cells to Th1-type immune responses. DC activation and mixed lymphocyte reaction (MLR) studies were performed to evaluate the immunogenicity of CD40L-dSIV in vitro. Expression levels of CD80, CD86, HLA-DR, and CD54 on DCs transduced with the dSIV incorporating CD40L (CD40L-dSIV) were significantly higher than on those transduced with dSIV. Moreover, CD40L-dSIV-transduced DCs expressed up to 10-fold more IL-12 than dSIV-transduced DCs. CD40L-dSIV-transduced DCs enhanced proliferation and gamma interferon secretion by naive T cells in an MLR. In addition, CD40L-dSIV-immunized mice exhibited stronger humoral and cell-mediated immune responses than dSIV-vaccinated animals. The results show that incorporating CD40L into the dSIV envelope significantly enhances immunogenicity. As a result, CD40L-dSIVs can be strong candidates for development of a safe and highly immunogenic AIDS vaccine.
PMCID: PMC2620918  PMID: 19036823
21.  Positive feedback regulation of maize NADPH oxidase by mitogen-activated protein kinase cascade in abscisic acid signalling 
Journal of Experimental Botany  2009;60(11):3221-3238.
In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling.
PMCID: PMC2718220  PMID: 19592501
Abscisic acid; feedback regulation; hydrogen peroxide; mitogen-activated protein kinase; NADPH oxidase; signal transduction; Zea mays
22.  Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Expressing Gamma Interferon Augment T-Cell Priming Responses In Vitro▿  
Journal of Virology  2006;81(5):2187-2195.
To increase the safety and efficacy of human immunodeficiency virus vaccines, several groups have conducted studies using the macaque model with single-cycle replicating simian immunodeficiency viruses (SIVs). However, these constructs had poor or diminished efficacy compared to live attenuated vaccines. We previously showed that immunization of macaques with live attenuated SIV with a deletion in the nef gene and expressing gamma interferon (IFN-γ) results in significantly enhanced safety and efficacy. To further enhance safety, we constructed and characterized single-cycle SIVs, pseudotyped with the glycoprotein of vesicular stomatitis virus, expressing different levels of macaque IFN-γ. Expression of IFN-γ did not alter the infectivity or antigenicity of pseudotyped SIV. The transduction of dendritic cells (DCs) by IFN-γ-expressing particles resulted in the up-regulation of costimulatory and major histocompatibility complex molecules. Furthermore, T cells primed with DCs transduced by SIV particles expressing high levels of IFN-γ and then stimulated with SIV induced significantly higher numbers of spot-forming cells in an enzyme-linked immunospot assay than did T cells primed with DCs transduced with SIV particles lacking the cytokine. In conclusion, we demonstrated that the transduction of DCs in vitro with pseudotyped single-cycle SIVs expressing IFN-γ increased DC activation and augmented T-cell priming activity.
PMCID: PMC1865962  PMID: 17166912
23.  Fly-DPI: database of protein interactomes for D. melanogaster in the approach of systems biology 
BMC Bioinformatics  2006;7(Suppl 5):S18.
Proteins control and mediate many biological activities of cells by interacting with other protein partners. This work presents a statistical model to predict protein interaction networks of Drosophila melanogaster based on insight into domain interactions.
Three high-throughput yeast two-hybrid experiments and the collection in FlyBase were used as our starting datasets. The co-occurrences of domains in these interactive events are converted into a probability score of domain-domain interaction. These scores are used to infer putative interaction among all available open reading frames (ORFs) of fruit fly. Additionally, the likelihood function is used to estimate all potential protein-protein interactions.
All parameters are successfully iterated and MLE is obtained for each pair of domains. Additionally, the maximized likelihood reaches its converged criteria and maintains the probability stable. The hybrid model achieves a high specificity with a loss of sensitivity, suggesting that the model may possess major features of protein-protein interactions. Several putative interactions predicted by the proposed hybrid model are supported by literatures, while experimental data with a low probability score indicate an uncertain reliability and require further proof of interaction.
Fly-DPI is the online database used to present this work. It is an integrated proteomics tool with comprehensive protein annotation information from major databases as well as an effective means of predicting protein-protein interactions. As a novel search strategy, the ping-pong search is a naïve path map between two chosen proteins based on pre-computed shortest paths. Adopting effective filtering strategies will facilitate researchers in depicting the bird's eye view of the network of interest. Fly-DPI can be accessed at .
This work provides two reference systems, statistical and biological, to evaluate the reliability of protein interaction. First, the hybrid model statistically estimates both experimental and predicted protein interaction relationships. Second, the biological information for filtering and annotation itself is a strong indicator for the reliability of protein-protein interaction. The space-temporal or stage-specific expression patterns of genes are also critical for identifying proteins involved in a particular situation.
PMCID: PMC1764474  PMID: 17254302
24.  POWER: PhylOgenetic WEb Repeater—an integrated and user-optimized framework for biomolecular phylogenetic analysis 
Nucleic Acids Research  2005;33(Web Server issue):W553-W556.
POWER, the PhylOgenetic WEb Repeater, is a web-based service designed to perform user-friendly pipeline phylogenetic analysis. POWER uses an open-source LAMP structure and infers genetic distances and phylogenetic relationships using well-established algorithms (ClustalW and PHYLIP). POWER incorporates a novel tree builder based on the GD library to generate a high-quality tree topology according to the calculated result. POWER accepts either raw sequences in FASTA format or user-uploaded alignment output files. Through a user-friendly web interface, users can sketch a tree effortlessly in multiple steps. After a tree has been generated, users can freely set and modify parameters, select tree building algorithms, refine sequence alignments or edit the tree topology. All the information related to input sequences and the processing history is logged and downloadable for the user's reference. Furthermore, iterative tree construction can be performed by adding sequences to, or removing them from, a previously submitted job. POWER is accessible at .
PMCID: PMC1160254  PMID: 15980533

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