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author:("Sun, liaodong")
2.  Interruption of nuclear localization of ATBF1 during the histopathologic progression of head and neck squamous cell carcinoma 
Head & neck  2012;35(7):1007-1014.
Background
The AT-motif Binding Factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers including head and neck squamous cell carcinomas (HNSCCs).
Methods
ATBF1 expression and localization were examined in five cell lines and 197 clinical specimens of HNSCC, and correlated with pathological and clinical characteristics.
Results
ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p=0.0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p<0.0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines.
Conclusions
Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.
doi:10.1002/hed.23077
PMCID: PMC3978689  PMID: 22791392
HNSCC; ATBF1; mislocalization; survival; tumor suppressor
3.  Parkin deficiency contributes to pancreatic tumorigenesis by inducing spindle multipolarity and misorientation 
Cell Cycle  2013;12(7):1133-1141.
Parkin, an E3 ubiquitin ligase well known for its role in the pathogenesis of juvenile Parkinson disease, has been considered as a candidate tumor suppressor in certain types of cancer. It remains unknown whether parkin is involved in the development of pancreatic cancer, the fourth leading cause of cancer-related deaths worldwide. Herein, we demonstrate the downregulation and copy number loss of the parkin gene in human pancreatic cancer specimens. The expression of parkin negatively correlates with clinicopathological parameters indicating the malignancy of pancreatic cancer. In addition, knockdown of parkin expression promotes the proliferation and tumorigenic properties of pancreatic cancer cells both in vitro and in mice. We further find that parkin deficiency increases the proportion of cells with spindle multipolarity and multinucleation. Parkin-depleted cells also show a significant increase in spindle misorientation. These findings indicate crucial involvement of parkin deficiency in the pathogenesis of pancreatic cancer.
doi:10.4161/cc.24215
PMCID: PMC3646869  PMID: 23470638
parkin; pancreatic cancer; cell proliferation; spindle multipolarity; spindle misorientation
4.  Characterization of Nuclear Localization and SUMOylation of the ATBF1 Transcription Factor in Epithelial Cells 
PLoS ONE  2014;9(3):e92746.
ATBF1/ZFHX3 is a large transcription factor that functions in development, tumorigenesis and other biological processes. ATBF1 is normally localized in the nucleus, but is often mislocalized in the cytoplasm in cancer cells. The mechanism underlying the mislocalization of ATBF1 is unknown. In this study, we analyzed the nuclear localization of ATBF1, and found that ectopically expressed ATBF1 formed nuclear body (NB)-like dots in the nucleus, some of which indeed physically associated with promyelocytic leukemia (PML) NBs. We also defined a 3-amino acid motif, KRK2615-2617, as the nuclear localization signal (NLS) for ATBF1. Interestingly, diffusely distributed nuclear SUMO1 proteins were sequestered into ATBF1 dots, which could be related to ATBF1's physical association with PML NBs, known SUMOylation hotspots. Furthermore, ATBF1 itself was SUMOylated. ATBF1 SUMOylation occurred at more than 3 lysine residues including K2349, K2806 and K3258 and was nuclear specific. Finally, the PIAS3 SUMO1 E3 ligase, which interacts with ATBF1 directly, diminished rather than enhanced ATBF1 SUMOylation, preventing the co-localization of ATBF1 with SUMO1 in the nucleus. These findings suggest that nuclear localization and SUMOylation are important for the transcription factor function of ATBF1, and that ATBF1 could cooperate with PML NBs to regulate protein SUMOylation in different biological processes.
doi:10.1371/journal.pone.0092746
PMCID: PMC3961433  PMID: 24651376
5.  Histone deacetylase 6 and cytoplasmic linker protein 170 function together to regulate the motility of pancreatic cancer cells 
Protein & Cell  2014;5(3):214-223.
Pancreatic cancer is a devastating disease with the worst prognosis among all the major human malignancies. The propensity to rapidly metastasize contributes significantly to the highly aggressive feature of pancreatic cancer. The molecular mechanisms underlying this remain elusive, and proteins involved in the control of pancreatic cancer cell motility are not fully characterized. In this study, we find that histone deacetylase 6 (HDAC6), a member of the class II HDAC family, is highly expressed at both protein and mRNA levels in human pancreatic cancer tissues. HDAC6 does not obviously affect pancreatic cancer cell proliferation or cell cycle progression. Instead, it significantly promotes the motility of pancreatic cancer cells. Further studies reveal that HDAC6 interacts with cytoplasmic linker protein 170 (CLIP-170) and that these two proteins function together to stimulate the migration of pancreatic cancer cells. These findings provide mechanistic insight into the progression of pancreatic cancer and suggest HDAC6 as a potential target for the management of this malignancy.
doi:10.1007/s13238-013-0010-3
PMCID: PMC3967059  PMID: 24474193
pancreatic cancer; cell motility; cell migration; cell proliferation; cell cycle
6.  Histone deacetylase 6 and cytoplasmic linker protein 170 function together to regulate the motility of pancreatic cancer cells 
Protein & Cell  2014;5(3):214-223.
Pancreatic cancer is a devastating disease with the worst prognosis among all the major human malignancies. The propensity to rapidly metastasize contributes significantly to the highly aggressive feature of pancreatic cancer. The molecular mechanisms underlying this remain elusive, and proteins involved in the control of pancreatic cancer cell motility are not fully characterized. In this study, we find that histone deacetylase 6 (HDAC6), a member of the class II HDAC family, is highly expressed at both protein and mRNA levels in human pancreatic cancer tissues. HDAC6 does not obviously affect pancreatic cancer cell proliferation or cell cycle progression. Instead, it significantly promotes the motility of pancreatic cancer cells. Further studies reveal that HDAC6 interacts with cytoplasmic linker protein 170 (CLIP-170) and that these two proteins function together to stimulate the migration of pancreatic cancer cells. These findings provide mechanistic insight into the progression of pancreatic cancer and suggest HDAC6 as a potential target for the management of this malignancy.
doi:10.1007/s13238-013-0010-3
PMCID: PMC3967059  PMID: 24474193
pancreatic cancer; cell motility; cell migration; cell proliferation; cell cycle
7.  Estrogen-Induced Nongenomic Calcium Signaling Inhibits Lipopolysaccharide-Stimulated Tumor Necrosis Factor α Production in Macrophages 
PLoS ONE  2013;8(12):e83072.
Estrogen is traditionally thought to exert genomic actions through members of the nuclear receptor family. Here, we investigated the rapid nongenomic effects of 17β-estradiol (E2) on tumor necrosis factor α (TNF-α) production following lipopolysaccharide (LPS) stimulation in mouse bone marrow-derived macrophages (BMMs). We found that LPS induced TNF-α production in BMMs via phosphorylation of p38 mitogen-activated protein kinase (MAPK). E2 itself did not affect the MAPK pathway, although it attenuated LPS-induced TNF-α production through suppression of p38 MAPK activation. Recently, G protein-coupled receptor 30 (GPR30) was suggested to be a membrane estrogen receptor (mER) that can mediate nongenomic estradiol signaling. We found that BMMs expressed both intracellular estrogen receptors (iER) and mER GPR30. The specific GPR30 antagonist G-15 significantly blocked effects of estradiol on LPS-induced TNF-α production, whereas an iER antagonist did not. Moreover, E2 induced a rapid rise in intracellular free Ca2+ that was due to the influx of extracellular Ca2+ and was not inhibited by an iER antagonist or silencing of iER. Ca2+ influx was also induced by an impermeable E2 conjugated to BSA (E2-BSA), which has been used to investigate the nongenomic effects of estrogen. Consequently, Ca2+, a pivotal factor in E2-stimulated nongenomic action, was identified as the key mediator. The inhibitory effects of E2 on LPS-induced TNF-α production and p38 MAPK phosphorylation were dependent on E2-triggered Ca2+ influx because BAPTA, an intracellular Ca2+ chelator, prevented these effects. Taken together, these data indicate that E2 can down-regulate LPS-induced TNF-α production via blockade of p38 MAPK phosphorylation through the mER-mediated nongenomic Ca2+ signaling pathway in BMMs.
doi:10.1371/journal.pone.0083072
PMCID: PMC3871562  PMID: 24376635
8.  A Meta-Analysis of Anti-Vascular Endothelial Growth Factor Remedy for Macular Edema Secondary to Central Retinal Vein Occlusion 
PLoS ONE  2013;8(12):e82454.
Background
Central retinal vein occlusion (CRVO) associates with severe vision outcome and no proven beneficial treatment. Our meta-analysis intended to appraise the efficacy and safety of anti-vascular endothelial growth factor (anti-VEGF) agents in macular edema (ME) following CRVO.
Methods
Data were collected and analyzed by Review Manager 5.2.1. We employed a random-effects model to eliminate between-study heterogeneity. Nfs (called fail-safe number) was calculated to evaluate the publication bias.
Results
We included 5 trials consisting 323 cases and 281 controls. Primary outcomes showed that overall comparison of anti-VEGF agents with placebo control yielded a 374% and 136% increased tendency for a gain of 15 letters or more on Early Treatment Diabetic Retinopathy Study (ETDRS) chart (95% confidence interval [95% CI]: 2.43–9.23; P<0.00001; I2 = 59%, 95% CI: 1.60–3.49; P<0.0001; I2 = 0%, respectively) at 6 and 12 months. Secondary outcomes showed that a 90% and 77% decreased risk at 6 and 12 months for a loss of 15 letters or more. The overall mean difference showed a statistically significance in best-corrected visual acuity (BCVA) on each time point. However, changes of central retinal thickness (CRT) lost significance at 12 months after 6-month as-needed treatment. The incidence of adverse events (AEs) had no statistical difference between anti-VEGF and placebo groups. Subgroup analyses indicated that patients receiving Aflibercept got the highest tendency to gain 15 letters or more (OR = 9.78; 95% CI: 4.43–21.56; P<0.00001). Age controlled analysis suggested a weaken tendency of BCVA improvement in age over 50 (MD = 12.26; 95% CI: 7.55–16.98; P<0.00001). Subgroup analysis by clinical classification showed a strengthen difference of BCVA changes at 6 months in ischemic type (MD = 19.65 letters, 95% CI: 13.15 to 26.14 letters, P<0.00001).
Conclusions
Our results showed that anti-VEGF agents were superior to placebo in CRVO-ME treatment with no statistically significant AEs, especially in younger people and for ischemic type.
doi:10.1371/journal.pone.0082454
PMCID: PMC3871640  PMID: 24376538
9.  A Micromachined Pressure Sensor with Integrated Resonator Operating at Atmospheric Pressure 
Sensors (Basel, Switzerland)  2013;13(12):17006-17024.
A novel resonant pressure sensor with an improved micromechanical double-ended tuning fork resonator packaged in dry air at atmospheric pressure is presented. The resonator is electrostatically driven and capacitively detected, and the sensor is designed to realize a low cost resonant pressure sensor with medium accuracy. Various damping mechanisms in a resonator that is vibrating at atmospheric pressure are analyzed in detail, and a formula is developed to predict the overall quality factor. A trade-off has been reached between the quality factor, stress sensitivity and drive capability of the resonator. Furthermore, differential sense elements and the method of electromechanical amplitude modulation are used for capacitive detection to obtain a large signal-to-noise ratio. The prototype sensor chip is successfully fabricated using a micromachining process based on a commercially available silicon-on-insulator wafer and is hermetically encapsulated in a custom 16-pin Kovar package. Preliminary measurements show that the fundamental frequency of the resonant pressure sensor is approximately 34.55 kHz with a pressure sensitivity of 20.77 Hz/kPa. Over the full scale pressure range of 100–400 kPa and the whole temperature range of −20–60 °C, high quality factors from 1,146 to 1,772 are obtained. The characterization of the prototype sensor reveals the feasibility of a resonant pressure sensor packaged at atmospheric pressure.
doi:10.3390/s131217006
PMCID: PMC3892843  PMID: 24335728
pressure sensor; resonant sensor; micromachining; micro resonator; atmospheric pressure; quality factor
10.  Heterozygous deletion of Atbf1 by the Cre-loxP system in mice causes preweaning mortality 
Genesis (New York, N.Y. : 2000)  2012;50(11):819-827.
Summary
ATBF1 is a large nuclear protein that contains multiple zinc-finger motifs and four homeodomains. In mammals, ATBF1 regulates differentiation, and its mutation and/or downregulation is involved in tumorigenesis in several organs. To gain more insight into the physiological functions of ATBF1, we generated and validated a conditional allele of mouse Atbf1 in which exons 7 and 8 were flanked by loxP sites (Atbf1flox). Germline deletion of a single Atbf1 allele was achieved by breeding to EIIa-cre transgenic mice, and Atbf1 heterozygous mice displayed reduced body weight, preweaning mortality, increased cell proliferation, and attenuated cytokeratin 18 (CK18) expression, indicating haploinsufficiency of Atbf1. Floxed Atbf1 mice will help us understand such biological processes as neuronal differentiation and tumorigenesis.
doi:10.1002/dvg.22041
PMCID: PMC3443507  PMID: 22644989
Atbf1; Cre-loxP; conditional knockout; heterozygous; preweaning mortality
11.  Validation of Polo-like kinase 1 as a therapeutic target in pancreatic cancer cells 
Cancer Biology & Therapy  2012;13(12):1214-1220.
Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase and plays a critical role in mitosis. PLK1 has also been regarded as a valuable target for cancer treatment, and several PLK1 inhibitors are currently undergoing clinical investigations. In this study, our data show that the expression level of PLK1 is upregulated in human pancreatic cancer cells. Molecular modeling studies indicate that DMTC inhibits PLK1 activity through competitive displacement of ATP from its binding pocket. Our data further show that DMTC suppresses the proliferation of pancreatic cancer cells and induces the formation of multinucleated cells, ultimately resulting in apoptosis. In addition, combination index analysis demonstrates that DMTC acts synergistically with the chemotherapeutic drug gemcitabine in inhibiting the proliferation of pancreatic cancer cells. These results thus suggest a potential of using PLK1 inhibitors for the treatment of pancreatic cancer.
doi:10.4161/cbt.21412
PMCID: PMC3469479  PMID: 22892842
PLK1; PLK1 inhibitor; cell proliferation and apoptosis; gemcitabine; pancreatic cancer
12.  Estrogen causes ATBF1 protein degradation through the estrogen-responsive E3 ubiquitin ligase EFP 
The Biochemical journal  2012;444(3):581-590.
We previously revealed that tumor suppressor ATBF1 formed an autoregulatory feedback loop with estrogen-ERα signaling to regulate estrogen-dependent cell proliferation in breast cancer cells. In this loop, ATBF1 inhibits the function of estrogen-ERα signaling while ATBF1 protein levels are fine-tuned by estrogen-induced transcriptional upregulation as well as ubiquitin proteasome pathway (UPP)-mediated protein degradation. Here we show that the estrogen-responsive finger protein (EFP) is an E3 ubiquitin ligase mediating estrogen-induced ATBF1 protein degradation. Knockdown increases but overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in estrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumors, due to both as directly-upregulated ERα target gene products, the levels of ATBF1 protein are positively correlated with the levels of EFP protein. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for estrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and estrogen-ERα signaling and thus implicate ATBF1 in estrogen-dependent breast development and carcinogenesis.
doi:10.1042/BJ20111890
PMCID: PMC3754848  PMID: 22452784
Estrogen; ATBF1; Protein degradation; E3 ubiquitin ligase; EFP; Breast cancer
13.  Estrogen causes degradation of KLF5 by inducing the E3 ubiquitin ligase EFP in ER-positive breast cancer cells 
The Biochemical journal  2011;437(2):323-333.
Krüppel-like factor 5 (KLF5) is a multifunctional transcription factor involved in cell proliferation, differentiation and carcinogenesis. In addition to frequent inactivation in different types of human cancers including breast cancer, KLF5 has been identified as an essential co-factor for the TGF-β tumor suppressor. In our previous study demonstrating a negative regulation of ER (estrogen receptor alpha) function by KLF5 in breast cancer cells, we noticed that estrogen reduced the protein level of KLF5. In this study, we tested whether and how estrogen-ER signaling regulates KLF5 protein. We found that estrogen caused the degradation of KLF5 protein, and the degradation was sensitive to proteasome inhibitors but not other inhibitors. The estrogen-inducible E3 ligase EFP was identified as a key player in estrogen-mediated degradation of KLF5, as knockdown and over-expression of EFP increased and decreased KLF5 protein levels respectively, and the decrease continued even when protein synthesis was blocked. EFP-mediated degradation impaired the function of KLF5 in gene transcription. While only unubiquitinated EFP interacted with KLF5, overexpression of EFP appeared to prevent the ubiquitination of KLF5 while resulting in heavy ubiquitination of the E3 itself. Furthermore, ubiquitination of EFP interrupted its interaction with KLF5. Although the mechanism for how EFP degrades KLF5 remains to be determined, these results suggest that estrogen causes the degradation of KLF5 protein by inducing the expression of EFP in ER-positive breast cancer cells.
doi:10.1042/BJ20101388
PMCID: PMC3733548  PMID: 21542805
KLF5; EFP; estrogen; ER; breast cancer
14.  A Novel, Sensitive Assay for High-Throughput Molecular Detection of Plasmodia for Active Screening of Malaria for Elimination 
Journal of Clinical Microbiology  2013;51(1):125-130.
Although malaria remains one of the leading infectious diseases in the world, the decline in malaria transmission in some area makes it possible to consider elimination of the disease. As countries approach elimination, malaria diagnosis needs to change from diagnosing ill patients to actively detecting infections in all carriers, including asymptomatic and low-parasite-load patients. However, few of the current diagnostic methods have both the throughput and the sensitivity required. We adopted a sandwich RNA hybridization assay to detect genus Plasmodium 18S rRNA directly from whole-blood samples from Plasmodium falciparum and Plasmodium vivax patients without RNA isolation. We tested the assay with 202 febrile patients from areas where malaria is endemic, using 20 μl of each blood sample in a 96-well plate format with a 2-day enzyme-linked immunosorbent assay (ELISA)-like work flow. The results were compared with diagnoses obtained using microscopy, a rapid diagnostic test (RDT), and genus-specific real-time PCR. Our assay identified all 66 positive samples diagnosed by microscopy, including 49 poorly stored samples that underwent multiple freeze-thaw cycles due to resource limitation. The assay uncovered three false-negative samples by microscopy and four false-negative samples by RDT and agreed completely with real-time PCR diagnosis. There was no negative sample by our assay that would show a positive result when tested with other methods. The detection limit of our assay for P. falciparum was 0.04 parasite/μl. The assay's simple work flow, high throughput, and sensitivity make it suitable for active malaria screening.
doi:10.1128/JCM.02010-12
PMCID: PMC3536241  PMID: 23100347
15.  Different Expression Patterns and Functions of Acetylated and Unacetylated Klf5 in the Proliferation and Differentiation of Prostatic Epithelial Cells 
PLoS ONE  2013;8(6):e65538.
KLF5 is a basic transcription factor that regulates multiple biological processes. While it was identified as a putative tumor suppressor in prostate cancer, likely due to its function as an effector of TGF-β in the inhibition of cell proliferation, KLF5 is unacetylated and promotes cell proliferation in the absence of TGF-β. In this study, we evaluated the expression and function of KLF5 in prostatic epithelial homeostasis and tumorigenesis using mouse prostates and human prostate epithelial cells in 3-D culture. Histological and molecular analyses demonstrated that unacetylated-Klf5 was expressed in basal or undifferentiated cells, whereas acetylated-Klf5 was expressed primarily in luminal and/or differentiated cells. Androgen depletion via castration increased both the level of Klf5 expression and the number of Klf5-positive cells in the remaining prostate. Functionally, knockdown of KLF5 in the human RWPE-1 prostate cell line decreased the number of spheres formed in 3-D culture. In addition, knockout of Klf5 in prostate epithelial cells, mediated by probasin promoter-driven Cre expression, did not cause neoplasia but promoted cell proliferation and induced hyperplasia when one Klf5 allele was knocked out. Knockout of both Klf5 alleles however, caused apoptosis rather than cell proliferation in the epithelium. In castrated mice, knockout of Klf5 resulted in more severe shrinkage of the prostate. These results suggest that KLF5 plays a role in the proliferation and differentiation of prostatic epithelial cells, yet loss of KLF5 alone is insufficient to induce malignant transformation in epithelial cells.
doi:10.1371/journal.pone.0065538
PMCID: PMC3673967  PMID: 23755247
16.  RNA Interference of GADD153 Protects Photoreceptors from Endoplasmic Reticulum Stress-Mediated Apoptosis after Retinal Detachment 
PLoS ONE  2013;8(3):e59339.
Background
Apoptosis of photoreceptors plays a critical role in the vision loss caused by retinal detachment (RD). Pharmacologic inhibition of photoreceptor cell death may prevent RD. This study investigated the role of GADD153 that participates in endoplasmic reticulum (ER) stress-mediated apoptosis of photoreceptor cells after RD.
Methods
Retinal detachment was created in Wistar rats by subretinal injection of hyaluronic acid. The rats were then randomly divided into four groups: normal control group, RD group, GADD153 RNAi group and vehicle group. RNA interference of GADD153 was performed using short hairpin RNA (shRNA). Expressions of GADD153 mRNA and protein were examined by RT-PCR and Western blotting analysis, respectively. GADD153 protein distribution in the retinal cells was observed using immunofluorescence confocal laser scanning microscopy. Apoptosis of retinal cells was determined by TdT-mediated fluorescein-16-dUTP nick-end labeling (TUNEL) assay.
Results
Lentivirus GADD153 shRNA with the most effective silencing effect was chosen for in vivo animal study and was successfully delivered into the retinal tissues. GADD153 mRNA and protein expressions in GADD153 RNAi group were significantly lower than those in the RD group. Silencing of GADD153 by RNAi protected photoreceptors from ER stress-induced apoptosis.
Conclusion
ER stress-mediated pathway is involved in photoreceptor cell apoptosis after RD. GADD153 is a key regulatory molecule regulating ER-stress pathways and plays a crucial role in the apoptosis of photoreceptor cells after RD.
doi:10.1371/journal.pone.0059339
PMCID: PMC3612068  PMID: 23555658
17.  Anti-folate combination therapies and their effect on the development of drug resistance in Plasmodium vivax 
Scientific Reports  2013;3:1008.
Can we predict the rise and spread of resistance to multi-drug therapy in a more predictable manner? We raise this question after analyzing over 500 Plasmodium vivax isolates collected from different, geographically isolated regions of China for sequence variation in and around the dhfr and dhps genes. We find: that resistance lineages have arisen at least once in each region; that there appears to have been little movement of parasite populations between these areas; and that highly resistant parasites contain dhfr and dhps alleles that are in linkage disequilibrium. We show a direct relationship between this linkage disequilibrium and a parasite's fitness in the absence of drug pressure. Such fitness would increase the spread of drug resistant phenotypes and is thus a selectable trait. These conclusions raise questions about the appropriate use of some other drug combinations to prevent and treat infection.
doi:10.1038/srep01008
PMCID: PMC3538286  PMID: 23301149
18.  Serum Fetuin-A Levels Related with Microalbuminuria in Diet-Induced Obese Rats 
BioMed Research International  2012;2013:795103.
The aim of the study was to investigate the association between elevated serum fetuin-A and increased urine albumin excretion in obese rats, and whether increased urine albumin excretion was modified by improving hepatic steatosis and lipid metabolism disorder. Male Wistar rats 4 weeks in age were randomly divided into three groups and fed with normal chow (control group), high-fat chow (obesity group), or high-fat chow plus fenofibrate (fenofibrate group). After 24 weeks, both body weight and visceral fat/body weight ratio in obese rats were higher than in controls. A difference in serology markers and pathology associated with hepatic steatosis was also found among the three groups. Serum fetuin-A and the expression of NF-κB in the liver were increased, while serum adiponectin was decreased in obese rats in comparison to controls (P < 0.01). Urinary albumin/creatinine ratio (ACR) was increased in the obesity group compared to controls (P < 0.01). The fenofibrate intervention reduced serum fetuin-A and NF-κB expression in the liver and increased serum adiponectin compared to obese rats and was accompanied by decrease in ACR. A positive correlation was found between ACR and fetuin-A (r = 0.602, P < 0.01), and a negative correlation was found between ACR and adiponectin (r = −0.635, P < 0.01). We conclude that elevated fetuin-A levels are associated with microalbuminuria in obese rats, and abnormal albuminuria is reversible by improving hepatic steatosis.
doi:10.1155/2013/795103
PMCID: PMC3591142  PMID: 23710462
19.  Atbf1 Regulates Pubertal Mammary Gland Development Likely by Inhibiting the Pro-Proliferative Function of Estrogen-ER Signaling 
PLoS ONE  2012;7(12):e51283.
ATBF1 is a candidate tumor suppressor that interacts with estrogen receptor (ER) to inhibit the function of estrogen-ER signaling in gene regulation and cell proliferation control in human breast cancer cells. We therefore tested whether Atbf1 and its interaction with ER modulate the development of pubertal mammary gland, where estrogen is the predominant steroid hormone. In an in vitro model of cell differentiation, i.e., MCF10A cells cultured in Matrigel, ATBF1 expression was significantly increased, and knockdown of ATBF1 inhibited acinus formation. During mouse mammary gland development, Atbf1 was expressed at varying levels at different stages, with higher levels during puberty, lower during pregnancy, and the highest during lactation. Knockout of Atbf1 at the onset of puberty enhanced ductal elongation and bifurcation and promoted cell proliferation in both ducts and terminal end buds of pubertal mammary glands. Enhanced cell proliferation primarily occurred in ER-positive cells and was accompanied by increased expression of ER target genes. Furthermore, inactivation of Atbf1 reduced the expression of basal cell markers (CK5, CK14 and CD44) but not luminal cell markers. These findings indicate that Atbf1 plays a role in the development of pubertal mammary gland likely by modulating the function of estrogen-ER signaling in luminal cells and by modulating gene expression in basal cells.
doi:10.1371/journal.pone.0051283
PMCID: PMC3520988  PMID: 23251482
20.  Therapeutic efficacy of artesunate in the treatment of uncomplicated Plasmodium falciparum malaria and anti-malarial, drug-resistance marker polymorphisms in populations near the China-Myanmar border 
Malaria Journal  2012;11:278.
Background
The aim of this study was to evaluate the clinical outcome after seven-day artesunate monotherapy for uncomplicated Plasmodium falciparum malaria in Yingjiang County along the China-Myanmar border and investigate genetic polymorphisms in the P. falciparum chloroquine-resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes.
Methods
Patients ≥ one year of age with fever (axillary temperature ≥37.5°C) or history of fever and P. falciparum mono-infection were included. Patients received anti-malarial treatment with artesunate (total dose of 16 mg/kg over seven days) by directly observed therapy. After a 28-day follow-up, treatment efficacy and effectiveness were assessed based on clinical and parasitological outcomes. Treatment failure was defined as recrudescence of the original parasite and distinguished with new infection confirmed by PCR. Analysis of gene mutation and amplification were performed by nested polymerase chain reaction.
Results
Sixty-five patients were enrolled; 10 withdrew from the study, and six were lost to follow-up. All but two patients demonstrated adequate clinical and parasitological response; 12 had detectable parasitaemia on day 3. These two patients were confirmed to be new infection by PCR. The efficacy of artesunate was 95.9%. The pfcrt mutation in codon 76 was found in all isolates (100%), and mutations in codons 71 and 72 were found in 4.8% of parasite isolates. No mutation of pfmdr1 (codons 86 or 1246) was found. Among all samples, 5.1% were wild type for pfdhfr, whereas the other samples had mutations in four codons (51, 59, 108 and 164), and mutations in pfdhps (codons 436, 437, 540 and 581) were found in all isolates. No samples had mutations in pfatp6 codons 623 or 769, but two new mutations (N683K and R756K) were found in 4.6% and 9.2% of parasite isolates, respectively.
Conclusion
Plasmodium falciparum infection was associated with slow parasite clearance and suspected artemisinin resistance at the China-Myanmar border area. The prevalence of pfcrt 76 T and markers for SP resistance are still high. It should be strengthened further on parasite clearance time or clearance half life to confirm the resistance status, and molecular epidemiology should provide complementary information to assess the appropriateness of current policies based on artemisinin derivatives.
doi:10.1186/1475-2875-11-278
PMCID: PMC3492106  PMID: 22898135
Molecular markers; Artesunate; Plasmodium falciparum
21.  Hypersensitive to Red and Blue 1 and Its Modification by Protein Phosphatase 7 Are Implicated in the Control of Arabidopsis Stomatal Aperture 
PLoS Genetics  2012;8(5):e1002674.
The stomatal pores are located on the plant leaf epidermis and regulate CO2 uptake for photosynthesis and the loss of water by transpiration. Their stomatal aperture therefore affects photosynthesis, water use efficiency, and agricultural crop yields. Blue light, one of the environmental signals that regulates the plant stomatal aperture, is perceived by the blue/UV-A light-absorbing cryptochromes and phototropins. The signal transduction cascades that link the perception of light to the stomatal opening response are still largely unknown. Here, we report two new players, Hypersensitive to Red and Blue 1 (HRB1) and Protein Phosphatase 7 (PP7), and their genetic and biochemical interactions in the control of stomatal aperture. Mutations in either HRB1 or PP7 lead to the misregulation of the stomatal aperture and reduce water loss under blue light. Both HRB1 and PP7 are expressed in the guard cells in response to a light-to-dark or dark-to-light transition. HRB1 interacts with PP7 through its N-terminal ZZ-type zinc finger motif and requires a functional PP7 for its stomatal opening response. HRB1 is phosphorylated in vivo, and PP7 can dephosphorylate HRB1. HRB1 is mostly dephosphorylated in a protein complex of 193 kDa in the dark, and blue light increases complex size to 285 kDa. In the pp7 mutant, this size shift is impaired, and HRB1 is predominately phosphorylated. We propose that a modification of HRB1 by PP7 under blue light is essential to acquire a proper conformation or to bring in new components for the assembly of a functional HRB1 protein complex. Guard cells control stomatal opening in response to multiple environmental or biotic stimuli. This study may furnish strategies that allow plants to enjoy the advantages of both constitutive and ABA-induced protection under water-limiting conditions.
Author Summary
Stomatal aperture is regulated by many environmental and biotic cues such as blue light, drought, elevated CO2 concentrations, high humidity, and pathogenic elicitors. Stomatal apertures vary over diurnal cycles, and stomata tend to be open during the day in response to blue light and tend to be closed at night. The blue/UV-A light-absorbing cryptochromes and phototropins are the receptors for the blue light response. We report the action of HRB1, a nuclear ZZ-type zinc finger protein, and PP7, a positive regulator of blue light signaling in the nucleus, in the signal transduction cascades downstream of blue light perception. Both hrb1 and pp7 mutants are more resistant to dehydration and show reductions in both water loss and blue light-regulated stomatal aperture. Our studies on their genetic and biochemical interactions offer novel insights on the network structure of the light signaling machinery and plant interactions with the environment. Periodic drought is one of the major environmental factors that limits biomass production and crop yield in a changing global climate. Our studies may open new possibilities to engineer plants to survive desiccation.
doi:10.1371/journal.pgen.1002674
PMCID: PMC3349726  PMID: 22589732
22.  Chromodomain helicase DNA binding protein 5 plays a tumor suppressor role in human breast cancer 
Introduction
The chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a tumor suppressor in a mouse model. The CHD5 locus at 1p36 is deleted, and its mutation has been detected in breast cancer. We, therefore, evaluated whether CHD5 plays a role in human breast cancer.
Methods
We screened mutations in 55 tumors, determined promoter methylation in 39 tumors, measured RNA expression in 90 tumors, analyzed protein expression in 289 tumors, and correlated expression changes with clinicopathological characteristics of breast cancer. Functional effects of CHD5 on cell proliferation, invasion and tumorigenesis were also tested.
Results
Although only one mutation was detected, CHD5 mRNA expression was significantly reduced, accompanied by frequent genomic deletion and promoter methylation, in breast cancer. The extent of methylation was significantly associated with reduced mRNA expression, and demethylating treatment restored CHD5 expression. Lower CHD5 mRNA levels correlated with lymph node metastasis (P = 0.026). CHD5 protein expression was also reduced in breast cancer, and lack of CHD5 expression significantly correlated with higher tumor stage, ER/PR-negativity, HER2 positivity, distant metastasis and worse patient survival (P ≤ 0.01). Functionally, ectopic expression of CHD5 in breast cancer cells inhibited cell proliferation and invasion in vitro and tumorigenesis in nude mice. Consistent with the inhibition of invasion, CHD5 down-regulated mesenchymal markers vimentin, N-cadherin and ZEB1 in breast cancer cells.
Conclusion
Down-regulation of CHD5, mediated at least in part by promoter methylation, contributes to the development and progression of human breast cancer.
doi:10.1186/bcr3182
PMCID: PMC3446335  PMID: 22569290
23.  Regulation of Vascular Endothelial Cell Polarization and Migration by Hsp70/Hsp90-Organizing Protein 
PLoS ONE  2012;7(4):e36389.
Hsp70/Hsp90-organizing protein (HOP) is a member of the co-chaperone family, which directly binds to chaperones to regulate their activities. The participation of HOP in cell motility and endothelial cell functions remains largely unknown. In this study, we demonstrate that HOP is critically involved in endothelial cell migration and angiogenesis. Tube formation and capillary sprouting experiments reveal that depletion of HOP expression significantly inhibits vessel formation from endothelial cells. Wound healing and transwell migration assays show that HOP is important for endothelial cell migration. By examination of centrosome reorientation and membrane ruffle dynamics, we find that HOP plays a crucial role in the establishment of cell polarity in response to migratory stimulus. Furthermore, our data show that HOP interacts with tubulin and colocalizes with microtubules in endothelial cells. These findings indicate HOP as a novel regulator of angiogenesis that functions through promoting vascular endothelial cell polarization and migration.
doi:10.1371/journal.pone.0036389
PMCID: PMC3340350  PMID: 22558459
24.  Single Endemic Genotype of Measles Virus Continuously Circulating in China for at Least 16 Years 
PLoS ONE  2012;7(4):e34401.
The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV) isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%–100% and 84.7%–100%, H1b were 97.1%–100% and 95.3%–100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR). Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years.
doi:10.1371/journal.pone.0034401
PMCID: PMC3332093  PMID: 22532829
25.  Characterization of the Duffy-Binding-Like Domain of Plasmodium falciparum Blood-Stage Antigen 332 
Malaria Research and Treatment  2011;2011:671439.
Studies on Pf332, a major Plasmodium falciparum blood-stage antigen, have largely been hampered by the cross-reactive nature of antibodies generated against the molecule due to its high content of repeats, which are present in other malaria antigens. We previously reported the identification of a conserved domain in Pf332 with a high degree of similarity to the Duffy-binding-like (DBL) domains of the erythrocyte-binding-like (EBL) family. We here describe that antibodies towards Pf332-DBL are induced after repeated exposure to P. falciparum and that they are acquired early in life in areas of intense malaria transmission. Furthermore, a homology model of Pf332-DBL was found to be similar to the structure of the EBL-DBLs. Despite their similarities, antibodies towards Pf332-DBL did not display any cross-reactivity with EBL-proteins as demonstrated by immunofluorescence microscopy, Western blotting, and peptide microarray. Thus the DBL domain is an attractive region to use in further studies on the giant Pf332 molecule.
doi:10.4061/2011/671439
PMCID: PMC3269649  PMID: 22312570

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