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1.  Motor preparation attenuates neural variability and beta-band LFP in parietal cortex 
Scientific Reports  2014;4:6809.
Accumulative evidence shows that neural variability is meaningful and powerful during brain information processing, but how cognitive state influences neural variability is still unclear. We studied neural variability during motor preparation in lateral intraparietal area (LIP), a brain area closely involved in saccade generation. During motor preparation, we observed significant variability decline, and the decline highly correlated with beta-band local field potential (LFP) fluctuations. Furthermore, we found similar variance-LFP correlations in both the memory-guided saccade task and the visually-guided saccade task. These results indicate a possible linkage between beta-band LFP and trial-to-trial neural variability.
PMCID: PMC4210872  PMID: 25348502
2.  Diagnosis of 25 genotypes of human papillomaviruses for their physical statuses in cervical precancerous/cancerous lesions: a comparison of E2/E6E7 ratio-based vs. multiple E1-L1/E6E7 ratio-based detection techniques 
Cervical lesions caused by integrated human papillomavirus (HPV) infection are highly dangerous because they can quickly develop into invasive cancers. However, clinicians are currently hampered by the lack of a quick, convenient and precise technique to detect integrated/mixed infections of various genotypes of HPVs in the cervix. This study aimed to develop a practical tool to determine the physical status of different HPVs and evaluate its clinical significance.
The target population comprised 1162 women with an HPV infection history of > six months and an abnormal cervical cytological finding. The multiple E1-L1/E6E7 ratio analysis, a novel technique, was developed based on determining the ratios of E1/E6E7, E2/E6E7, E4E5/E6E7, L2/E6E7 and L1/E6E7 within the viral genome. Any imbalanced ratios indicate integration. Its diagnostic and predictive performances were compared with those of E2/E6E7 ratio analysis. The detection accuracy of both techniques was evaluated using the gold-standard technique “detection of integrated papillomavirus sequences” (DIPS). To realize a multigenotypic detection goal, a primer and probe library was established.
The integration rate of a particular genotype of HPV was correlated with its tumorigenic potential and women with higher lesion grades often carried lower viral loads. The E1-L1/E6E7 ratio analysis achieved 92.7% sensitivity and 99.0% specificity in detecting HPV integration, while the E2/E6E7 ratio analysis showed a much lower sensitivity (75.6%) and a similar specificity (99.3%). Interference due to episomal copies was observed in both techniques, leading to false-negative results. However, some positive results of E1-L1/E6E7 ratio analysis were missed by DIPS due to its stochastic detection nature. The E1-L1/E6E7 ratio analysis is more efficient than E2/E6E7 ratio analysis and DIPS in predicting precancerous/cancerous lesions, in which both positive predictive values (36.7%-82.3%) and negative predictive values (75.9%-100%) were highest (based on the results of three rounds of biopsies).
The multiple E1-L1/E6E7 ratio analysis is more sensitive and predictive than E2/E6E7 ratio analysis as a triage test for detecting HPV integration. It can effectively narrow the range of candidates for colposcopic examination and cervical biopsy, thereby lowering the expense of cervical cancer prevention.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0282-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4192431  PMID: 25269554
Human papillomavirus; Physical status; Integration; E1; E2; E6; E7; L1; Polymerase chain reaction; Cervical cancer
3.  AKR1C3 as a Target in Castrate Resistant Prostate Cancer 
Aberrant androgen receptor (AR) activation is the major driver of castrate resistant prostate cancer (CRPC). CRPC is ultimately fatal and more therapeutic agents are needed to treat this disease. Compounds that target the androgen axis by inhibiting androgen biosynthesis and or AR signaling are potential candidates for use in CRPC treatment and are currently being pursued aggressively. Aldo-keto reductase 1C3 (AKR1C3) plays a pivotal role in androgen biosynthesis within the prostate. It catalyzes the 17-ketoreduction of weak androgen precursors to give testosterone and 5α-dihydrotestosterone. AKR1C3 expression and activity has been implicated in the development of CRPC, making it a rational target. Selective inhibition of AKR1C3 will be important however, due to the presence of closely related isoforms, AKR1C1 and AKR1C2 that are also involved in androgen inactivation. We examine the evidence that supports the vital role of AKR1C3 in CRPC and recent developments in the discovery of potent and selective AKR1C3 inhibitors.
PMCID: PMC3805777  PMID: 23748150
Type 5 17β-hydroxysteroid dehydrogenase; prostaglandin F synthase; prostate cancer; androgens; nonsteroidal anti-inflammatory drugs
4.  Characterization of Murine Pituitary-Derived Cell Lines Tpit/F1, Tpit/E and TtT/GF 
The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.
PMCID: PMC4139504  PMID: 24881870
Differentiation; Pituitary cell line; Stem/progenitor cell; SOX2
5.  Competitive Stem Cell Recruitment by Multiple Cytotactic Cues 
Lab on a chip  2013;13(6):1156-1164.
A multitude of cytotactic cues direct cell migration in development, cancer metastasis and wound healing. However, our understanding of cell motility remains fragmented partially because current migration devices only allow the study of independent factors. We developed a cell motility assay that allows competitive recruitment of a given cell population simultaneously by gradients of multiple cytotactic cues, observable under real-time imaging. Well-defined uniform gradients of cytotactic cues can be independently generated and sustained in each channel. As a case study, bone marrow mesenchymal stem/stromal cells (MSCs) were exposed to 15 cytokines that are commonly present in arthritis. Cytokines that induced robust recruitment of MSCs in multiple groups were selected to ‘compete’ in a final round to yield the most chemotactic factor(s) based on cell migration numbers, distances, migration indices and motility over time. The potency of a given cytokine in competition frequently differed from its individual action, substantiating the need to test multiple cytokines concurrently due to synergistic or antagonistic effects. This new device has the rare capacity to screen molecules that induce cell migration in cancer therapy, drug development and tissue regeneration.
PMCID: PMC4093799  PMID: 23364311
Stem cells; migration; microfluidic; microfabrication; cell homing; cytokines
6.  Dentin and dental pulp regeneration by the patient’s endogenous cells 
Endodontic topics  2013;28(1):106-117.
The goal of regenerative endodontics is to restore the functions of the dental pulp–dentin complex. Two approaches are being applied toward dental pulp–dentin regeneration: cell transplantation and cell homing. The majority of previous approaches are based on cell transplantation by delivering ex vivo cultivated cells toward dental pulp or dentin regeneration. Many hurdles limit the clinical translation of cell transplantation such as the difficulty of acquiring and isolating viable cells, uncertainty of what cells or what fractions of cells to use, excessive cost of cell manipulation and transportation, and the risk of immune rejection, pathogen transmission, and tumorigenesis in associated with ex vivo cell manipulation. In contrast, cell homing relies on induced chemotaxis of endogenous cells and therefore circumvents many of the difficulties that are associated with cell transplantation. An array of proteins, peptides, and chemical compounds that are yet to be identified may orchestrate endogenous cells to regenerate dental pulp–dentin complex. Both cell transplantation and cell homing are scientifically valid approaches; however, cell homing offers a number of advantages that are compatible with the development of clinical therapies for dental pulp–dentin regeneration.
PMCID: PMC4070522  PMID: 24976816
7.  Expression of three essential antioxidants of Helicobacter pylori in clinical isolates*  
Objective: Helicobacter pylori maintains long-term persistence in the host and combats oxidative stress via many antioxidant proteins, which are expected to be relevant to bacterial-associated gastric diseases. We aimed to investigate the expression of three essential antioxidants in H. pylori strains isolated from patients with different clinical outcomes. Methods: Forty H. pylori strains were isolated from endoscopic biopsy specimens of gastric mucosa from 13 patients with gastric cancer, 13 with peptic ulcer, and 14 with gastritis. The expression of thioredoxin 1 (Trx1), arginase (RocF), and alkyl hydroperoxide reductase (AhpC) in H. pylori was measured by real-time PCR. Comparisons among multiple sample sets were analyzed using a one-way ANOVA test. Pearson’s correlation test was used to assess relationships among multiple continuous variables. Results: Trx1 expression of H. pylori in gastric cancer and peptic ulcer tissues was higher than that in tissues with gastritis. RocF expression of H. pylori in gastric cancer tissues was higher than that in tissues exhibiting peptic ulcer and gastritis. However, we did not find any differences in AhpC expression in samples from patients with different clinical outcomes. The expression of Trx1 and RocF had a positive, linear correlation. The expression of Trx1 and AhpC had a positive correlation without a linear trend. We found no correlation between the expression of RocF and AhpC. Conclusions: Our observations indicate that the expression of Trx1 and RocF in H. pylori might be related to gastric carcinogenesis. In H. pylori, the expression of members of the antioxidant system may be correlated and relevant to gastric cancer.
PMCID: PMC4076607  PMID: 24793768
Antioxidant; Gastric cancer; Helicobacter pylori; Oxidative stress
8.  Development of Potent and Selective Indomethacin Analogs for the Inhibition of AKR1C3 (Type 5 17β-Hydroxysteroid Dehydrogenase/Prostaglandin F Synthase) in Castrate-Resistant Prostate Cancer 
Journal of medicinal chemistry  2013;56(6):2429-2446.
Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogs, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP+ ·2′-des-methylindomethacin crystal structure was determined and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC.
PMCID: PMC3638264  PMID: 23432095
9.  Temperature and Cyanobacterial Bloom Biomass Influence Phosphorous Cycling in Eutrophic Lake Sediments 
PLoS ONE  2014;9(3):e93130.
Cyanobacterial blooms frequently occur in freshwater lakes, subsequently, substantial amounts of decaying cyanobacterial bloom biomass (CBB) settles onto the lake sediments where anaerobic mineralization reactions prevail. Coupled Fe/S cycling processes can influence the mobilization of phosphorus (P) in sediments, with high releases often resulting in eutrophication. To better understand eutrophication in Lake Taihu (PRC), we investigated the effects of CBB and temperature on phosphorus cycling in lake sediments. Results indicated that added CBB not only enhanced sedimentary iron reduction, but also resulted in a change from net sulfur oxidation to sulfate reduction, which jointly resulted in a spike of soluble Fe(II) and the formation of FeS/FeS2. Phosphate release was also enhanced with CBB amendment along with increases in reduced sulfur. Further release of phosphate was associated with increases in incubation temperature. In addition, CBB amendment resulted in a shift in P from the Fe-adsorbed P and the relatively unreactive Residual-P pools to the more reactive Al-adsorbed P, Ca-bound P and organic-P pools. Phosphorus cycling rates increased on addition of CBB and were higher at elevated temperatures, resulting in increased phosphorus release from sediments. These findings suggest that settling of CBB into sediments will likely increase the extent of eutrophication in aquatic environments and these processes will be magnified at higher temperatures.
PMCID: PMC3969358  PMID: 24682039
10.  Enhanced optical output power of blue light-emitting diodes with quasi-aligned gold nanoparticles 
The output power of the light from GaN-based light-emitting diodes (LEDs) was enhanced by fabricating gold (Au) nanoparticles on the surface of p-GaN. Quasi-aligned Au nanoparticle arrays were prepared by depositing Au thin film on an aligned suspended carbon nanotube thin film surface and then putting the Au-CNT system on the surface of p-GaN and thermally annealing the sample. The size and position of the Au nanoparticles were confined by the carbon nanotube framework, and no other additional residual Au was distributed on the surface of the p-GaN substrate. The output power of the light from the LEDs with Au nanoparticles was enhanced by 55.3% for an injected current of 100 mA with the electrical property unchanged compared with the conventional planar LEDs. The enhancement may originate from the surface plasmon effect and scattering effect of the Au nanoparticles.
PMCID: PMC3882480  PMID: 24393473
GaN; Light-emitting diodes (LEDs); Au nanoparticles; Carbon nanotube
11.  Characterization of Thermophilic Archaeal Isopentenyl Phosphate Kinases 
Biochemistry  2010;49(1):10.1021/bi9017957.
Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the essential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of the genomes of several members of the Archaea failed to identify genes for the enzymes required to convert phosphomevalonate (PM) to IPP in Eukaryotes. The recent discovery of an isopentenyl kinase (IPK) in Methanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM is decarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast search using the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycin resistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in a reaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in the search, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and the His-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze the reversible phosphorylation of IP by ATP, Keq = 6.3 ± 1. The catalytic efficiencies (V/K) of the proteins were ~2 × 106 M−1s−1. In the reverse direction, ADP was a substrate inhibitor for THA IPK, KiADP = 58 ± 6 µM but not for MTH IPK. Both enzymes were active over a broad range of pH and temperature. Five compounds, dimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, and geranyl phosphate, were evaluated as alternative substrate for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate.
PMCID: PMC3856865  PMID: 19928876
12.  Systemic mesenchymal stem cells reduce growth rate of cisplatin-resistant ovarian cancer 
Epithelial ovarian cancer is one of the most malignant cancers in women and resistant to chemotherapy is the major obstacle for the five-year survival rate. Cisplatin is one of the effective anticancer drug used in the ovarian cancer. To find a good strategy to cure the tumors which is resistant to cisplatin, the cisplatin-resistant 3SKOV3 cells were selected from SKOV-3 ovarian cancer cells. Furthermore, the isolated mesenchymal stem cells were infused systemically to try to cure the transplanted tumor induced by 3SKOV3 cells in nude mice. The morphology and cell membrane CD44 expression were investigated by microscope and flow cytometry. The biological behaviors of resistant 3SKOV3 and its parental SKOV3 cells, including proliferation, adhesion, and cell cycle were determined by CCK8, absorbance assay and FCM methods. The transplanted tumors were set up in nude mice with 3SKOV3 cells injection. The growth rate of transplanted tumors was detected following with MSCs injection. The 3SKOV3 cells have different morphologic manifestation and expressed high level of CD44 molecule. At the same time, 3SKOV3 cells have less adhesion ability and less S-phase ratio. The isolated MSCs from bone marrow could inhibit the growth of transplanted tumor via systemic injection. The cisplatin-resistant 3SKOV3 cells have the different biological behaviors as its parental SKOV3 cells. The present study indicated that systemic MSCs have the therapeutic role on ovarian cancer. However, further investigations are in progress to elucidate the underlying mechanism.
PMCID: PMC3816820  PMID: 24228113
Cisplatin; resistance; ovarian cancer; mesenchymal stem cells; nude mice
13.  Detection of Human Herpesviruses (HHVs) in Semen of Human Male Infertile Patients 
Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21–49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.
PMCID: PMC3934121  PMID: 23748714
Human herpesvirus (HHV); Human; Male infertility; Spermatogenesis; Thymidine kinase
14.  Which T Category of Nasopharyngeal Carcinoma May Benefit Most from Volumetric Modulated Arc Therapy Compared with Step and Shoot Intensity Modulated Radiation Therapy 
PLoS ONE  2013;8(9):e75304.
To compare volumetric modulated arc therapy (VMAT) with conventional step and shoot intensity modulated radiation therapy (s-IMRT) in nasopharyngeal carcinoma (NPC) patients, and identify which T category patient gains the maximum benefit from VMAT.
Fifty-two patients that randomly selected from 205 patients received VMAT at a single center were retrospectively replanned with s-IMRT. For a fair comparison, the planning target volume (PTV) coverage of the 2 plans was normalized to the same level. A standard planning constraint set was used; the constraints for the organs at risk (OARs) were individually adapted. The calculated doses to the PTV and OARs were compared for s-IMRT and VMAT plans generated using the Monaco treatment planning system.
VMAT and s-IMRT plans had similar PTV coverage and OAR sparing within all T categories. However, in stratified analysis, VMAT plans lead to better or similar sparing of the OARs in early T category patients; and lead to poorer sparing of the OARs in advanced T category patients (P<0.05). VMAT shows significant advantages for low dose burden (P<0.05) compared with s-IMRT. The delivery time per fraction for VMAT (424±64 s) was shorter than s-IMRT (778 ± 126 s, p<0.01).
VMAT provides similar dose coverage of the PTVs and similar/better normal tissue sparing in early T category NPC, and poorer OARs sparing in advanced T category NPC. And VMAT shows significant advantages for low dose burden and delivery time.
PMCID: PMC3783380  PMID: 24086503
15.  Inhibition of δ-opioid receptors induces brain glioma cell apoptosis through the mitochondrial and protein kinase C pathways 
Oncology Letters  2013;6(5):1351-1357.
Brain glioma is a malignant tumor with a high incidence rate and poor prognosis that has become a focus of studies of central nervous system diseases. Previous studies have suggested that δ-opioid receptors may affect the proliferation and apoptosis of numerous types of tumor cells. However, to date, their precise mechanism(s) of action have not been elucidated. The present study aimed to investigate the effects of inhibiting δ-opioid receptors in brain glioma cell proliferation and apoptosis and their relevant molecular mechanisms. Various doses of naltrindole were supplied to treat brain glioma cells using the MTT method to assess the proliferation index. Flow cytometry was used to investigate the changes in cell apoptosis and mitochondrial membrane potential. The expression levels of Bax, Bcl-2, Bcl-xL, cytochrome c, caspase-9, caspase-3 and protein kinase C (PKC) were measured using western blotting. Naltrindole was observed to inhibit brain glioma cell proliferation and promote apoptosis in a dose- and time-dependent manner. Furthermore, the addition of naltrindole lead to changes in the brain glioma cell membrane potential and regulated Bax translocation to the mitochondrial membrane, consequently promoting the release of cytochrome c into the cytoplasm, followed by the activation of caspase-9 and -3, which caused cell apoptosis. In addition, naltrindole was able to regulate the expression levels of the cellular internal phosphorylated PKC proteins, which are closely associated with the inhibition of cell proliferation. In conclusion, the inhibition of δ-opioid receptors may inhibit brain glioma cell proliferation and lead to apoptosis, which is closely associated with the mitochondrial and PKC pathways.
PMCID: PMC3813693  PMID: 24179523
δ-opioid receptor; glioma; apoptosis; mitochondria; protein kinase C pathway
16.  Association of Helicobacter pylori babA2 with peptic ulcer disease and gastric cancer 
AIM: To investigate the association between babA2 gene and peptic ulcer disease (PUD) and gastric cancer (GC) in Helicobacter pylori-infected populations.
METHODS: We evaluated the relationship between babA2 and clinical outcomes (PUD and GC) using a meta-analysis. A literature search was performed using the PubMed and Web of Science databases for relevant case-control studies that met the defined inclusion criteria. The ORs and 95%CIs were calculated to estimate the association between babA2 genotype and clinical outcomes. A fixed-effect or random-effect model was performed depending on the absence or presence of significant heterogeneity.
RESULTS: A total of 25 articles with 38 studies met the inclusion criteria and were finally included in this meta-analysis. The results showed that the babA2 genotype was significantly associated with an increased risk of PUD (OR = 2.069, 95%CI: 1.530-2.794, P < 0.001) and especially in the subgroup of duodenal ulcer (OR = 1.588, 95%CI: 1.141-2.209, P = 0.006). Moreover, a significant association between babA2 gene and PUD and duodenal ulcer (OR = 2.739, 95%CI: 1.860-4.032, P < 0.001; OR = 2.239, 95%CI: 1.468-3.415, P < 0.001, respectively) was observed in western countries but not in Asian countries.
CONCLUSION: We demonstrated that the presence of babA2 may be associated with increased risks for PUD, especially duodenal ulcer, in western countries.
PMCID: PMC3710429  PMID: 23864790
Helicobacter pylori; babA2; Peptic ulcer; Gastric cancer; Risk
17.  Radiation-Induced Temporal Lobe Injury for Nasopharyngeal Carcinoma: A Comparison of Intensity-Modulated Radiotherapy and Conventional Two-Dimensional Radiotherapy 
PLoS ONE  2013;8(7):e67488.
To compare the radiation-induced temporal lobe injury (TLI) in patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (IMRT) or two-dimensional conventional radiotherapy (2D-CRT).
Patients and Methods
1276 cases of NPC treated with IMRT or 2D-CRT were retrospectively reviewed. A diagnosis of TLI was made on follow-up magnetic resonance imaging (MRI).
The crude incidence of TLI was 7.5% and 10.8% (P = 0.048), and the actuarial 5-year incidence was 16% and 34.9% (P<0.001) for the IMRT and 2D-CRT groups, respectively. Multivariate analysis revealed both T stage (P<0.001) and radiation technique (P<0.001) as independent predictors. Patients with T1, T2 and T3 disease had a significantly higher risk when treated with 2D-CRT (P = 0.005, 0.016, <0.001, respectively). This trend was not evident for T4 patients (P = 0.680). The 2D-CRT group had a longer latency for the development of TLI (P<0.001). Those with T4 disease had a shorter median time to TLI (P = 0.006, 0.042, <0.001 when compared with T1, T2 and T3, respectively).
IMRT is superior to 2DRT for the management of T1-T3 NPC in terms of sparing the temporal lobe. The high incidence of TLI in T4 disease needs to be addressed.
PMCID: PMC3707870  PMID: 23874422
18.  Concentration-dependent control of pyruvate kinase M mutually exclusive splicing by hnRNP proteins 
Expression of the mammalian pyruvate kinase M (PKM) gene provides an important example of mutually exclusive splicing. We showed previously that the hnRNP proteins A1, A2 and PTB play a critical role in this process. Here we provide evidence that concentration-dependent interactions involving a network of these proteins are sufficient to determine the outcome of PKM splicing. At high concentrations, such as found in most cancer cells, hnRNP A1 binding to two sites in the upstream regulated exon (exon 9) orchestrates cooperative interactions leading to exon 9 exclusion. At lower concentrations, binding shifts to downstream intronic sites such that exon 9 is included and exon 10 largely excluded, with any mRNA including both exons degraded by nonsense-mediated decay. Together our results provide a mechanism by which a small number of general factors control alternative splicing of a widely expressed transcript.
PMCID: PMC3698866  PMID: 22307054
19.  Subcutaneous administration of rhIGF-I post irradiation exposure enhances hematopoietic recovery and survival in BALB/c mice 
Journal of radiation research  2012;53(4):581-587.
It is unclear how to effectively mitigate against irradiation injury. In this study, we studied the capacity of recombinant human insulin-like growth factor-I (rhIGF-I) on hematologic recovery in irradiated BALB/c mice and its possible mechanism. BALB/c mice were injected with rhIGF-I subcutaneously at a dose of 100 μg/kg twice daily for 7 days after total body irradiation. Compared with a saline control group, treatment with rhIGF-I significantly improved the survival of mice after lethal irradiation (7.5 Gy). It was found that treatment with rhIGF-I not only could increase the frequency of Sca-1+ cells in bone marrow harvested at Day 14 after irradiation, but also it could decrease the apoptosis of mononuclear cells induced by irradiation as measured by flow cytometry, suggesting that rhIGF-I may mediate its effects primarily through promoting hematopoietic stem cell/progenitor survival and protecting mononuclear cells from apoptosis after irradiation exposure. Moreover, we have found that rhIGF-I might facilitate thrombopoiesis in an indirect way. Our data demonstrated that rhIGF-I could promote overall hematopoietic recovery after ionizing radiation and reduce the mortality when administered immediately post lethal irradiation exposure.
PMCID: PMC3393355  PMID: 22843623
insulin-like growth factor-I; irradiation injury; hematopoietic recovery; apoptosis
21.  Crystal structures of AKR1C3 containing an N-(aryl)amino-benzoate inhibitor and a bifunctional AKR1C3 inhibitor and androgen receptor antagonist. Therapeutic leads for castrate resistant prostate cancer 
Castrate resistant prostate cancer (CRPC) is associated with increased androgen receptor (AR) signaling often brought about by elevated intratumoral androgen biosynthesis and AR amplification. Inhibition of androgen biosynthesis and/or AR antagonism should be efficacious in the treatment of CRPC. AKR1C3 catalyzes the formation of potent AR ligands from inactive precursors and is one of the most upregulated genes in CRPC. AKR1C3 inhibitors should not inhibit the related isoforms, AKR1C1 and AKR1C2 that are involved in 5α-dihydrotestosterone inactivation in the prostate. We have previously developed a series of flufenamic acid analogs as potent and selective AKR1C3 inhibitors (Adeniji A.O. et al., J Med Chem. 2012, 55, 2311). Here we report the X-ray crystal structures of one lead compound 3-((4′-(trifluoromethyl)phenyl) amino)benzoic acid (1) in complex with AKR1C3. Compound 1 adopts a similar binding orientation as flufenamic acid, however, its phenylamino ring projects deeper into a subpocket and confers selectivity over the other AKR1C isoforms. We exploited the observation that some flufenamic acid analogs also act as AR antagonists and synthesized a second generation inhibitor, 3-[4′-(nitronaphthalen-1-amino))benzoic acid (2). Compound 2 retained nanomolar potency and selective inhibition of AKR1C3 but also acted as an AR antagonist. It inhibited 5α-dihydrotestosterone stimulated AR reporter gene activity with an IC50 = 4.7 μM and produced a concentration dependent reduction in androgen receptor levels in prostate cancer cells. The in vitro and cell-based effects of compound 2 make it a promising lead for development of dual acting agent for CRPC. To illuminate the structural basis of AKR1C3 inhibition, we also report the crystal structure of the AKR1C3•NADP+•2 complex, which shows that compound 2 forms a unique double decker structure with AKR1C3.
PMCID: PMC3348334  PMID: 22507964
Aldo-keto reductase; non-steroidal anti-inflammatory drugs (NSAIDs); N-phenylanthranilic acids; adaptive androgen biosynthesis; androgen receptor antagonist; competitive inhibition
22.  Metabolism of the Synthetic Progestogen Norethynodrel by Human Ketosteroid Reductases of the Aldo-Keto Reductase Superfamily 
Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17β-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3β-hydroxy metabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3β-hydroxy NOR, while AKR1C3 gave 3β-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ4-isomer of NOR and the most commonly used synthetic progestin, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3β-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB.
PMCID: PMC3303946  PMID: 22210085
Ketosteroid reduction; progestin; oral contraceptive
23.  Development of Potent and Selective Inhibitors of Aldo-Keto Reductase 1C3 (type 5 17β-Hydroxysteroid Dehydrogenase) Based on N-Phenyl-Aminobenzoates and Their Structure Activity Relationships 
Journal of Medicinal Chemistry  2012;55(5):2311-2323.
Aldo-keto reductase 1C3 (AKR1C3; type 5 17β-hydroxysteroid dehydrogenase) is overexpressed in castrate resistant prostate cancer (CRPC) and is implicated in the intratumoral biosynthesis of testosterone and 5α-dihydrotestosterone. Selective AKR1C3 inhibitors are required since compounds should not inhibit the highly related AKR1C1 and AKR1C2 isoforms which are involved in the inactivation of 5α-dihydrotestosterone. NSAIDs, N-phenylanthranilates in particular are potent but non-selective AKR1C3 inhibitors. Using flufenamic acid, 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid as lead compound, five classes of structural analogs were synthesized and evaluated for AKR1C3 inhibitory potency and selectivity. Structure activity relationship (SAR) studies revealed that a meta-carboxylic acid group relative to the amine conferred pronounced AKR1C3 selectivity without loss of potency, while electron withdrawing groups on the phenylamino B-ring were optimal for AKR1C3 inhibition. Lead compounds did not inhibit COX-1 or COX-2 but blocked the AKR1C3 mediated production of testosterone in LNCaP-AKR1C3 cells. These compounds offer promising leads towards new therapeutics for CRPC.
PMCID: PMC3298089  PMID: 22263837
24.  Locoregional extension patterns of nasopharyngeal carcinoma and suggestions for clinical target volume delineation 
Chinese Journal of Cancer  2012;31(12):579-587.
Clinical target volume (CTV) delineation is crucial for tumor control and normal tissue protection. This study aimed to define the locoregional extension patterns of nasopharyngeal carcinoma (NPC) and to improve CTV delineation. Magnetic resonance imaging scans of 2366 newly diagnosed NPC patients were reviewed. According to incidence rates of tumor invasion, the anatomic sites surrounding the nasopharynx were classified into high-risk (>30%), medium-risk (5%–30%), and low-risk (<5%) groups. The lymph node (LN) level was determined according to the Radiation Therapy Oncology Group guidelines, which were further categorized into the upper neck (retropharyngeal region and level II), middle neck (levels III and Va), and lower neck (levels IV and Vb and the supraclavicular fossa). The high-risk anatomic sites were adjacent to the nasopharynx, whereas those at medium- or low-risk were separated from the nasopharynx. If the high-risk anatomic sites were involved, the rates of tumor invasion into the adjacent medium-risk sites increased; if not, the rates were significantly lower (P < 0.01). Among the 1920 (81.1%) patients with positive LN, the incidence rates of LN metastasis in the upper, middle, and lower neck were 99.6%, 30.2%, and 7.2%, respectively, and skip metastasis happened in only 1.2% of patients. In the 929 patients who had unilateral upper neck involvement, the rates of contralateral middle neck and lower neck involvement were 1.8% and 0.4%, respectively. Thus, local disease spreads stepwise from proximal sites to distal sites, and LN metastasis spreads from the upper neck to the lower neck. Individualized CTV delineation for NPC may be feasible.
PMCID: PMC3777458  PMID: 22854064
Nasopharyngeal carcinoma; magnetic resonance imaging; local extension; lymph node spread; clinical target volume
25.  High Levels of Aromatic Amino Acids in Gastric Juice during the Early Stages of Gastric Cancer Progression 
PLoS ONE  2012;7(11):e49434.
Early-stage gastric cancer is mostly asymptomatic and can easily be missed easily by conventional gastroscopy. Currently, there are no useful biomarkers for the early detection of gastric cancer, and their identification of biomarkers is urgently needed.
Gastric juice was obtained from 185 subjects that were divided into three groups: non-neoplastic gastric disease (NGD), advanced gastric cancer and early gastric cancer (EGC). The levels of aromatic amino acids in the gastric juice were quantitated using high-performance liquid chromatography.
The median values (25th to 75th percentile) of tyrosine, phenylalanine and tryptophan in the gastric juice were 3.8 (1.7–7.5) µg/ml, 5.3 (2.3–9.9) µg/ml and 1.0 (0.4–2.8) µg/ml in NGD; 19.4 (5.8–72.4) µg/ml, 24.6 (11.5–73.7) µg/ml and 8.3 (2.1–28.0) µg/ml in EGC. Higher levels of tyrosine, phenylalanine and tryptophan in the gastric juice were observed in individuals of EGC groups compared those of the NGD group (NGD vs. EGC, P<0.0001). For the detection of EGC, the areas under the receiver operating characteristic curves (AUCs) of each biomarker were as follows: tyrosine, 0.790 [95% confidence interval (CI), 0.703–0.877]; phenylalanine, 0.831 (95% CI, 0.750–0.911); and tryptophan, 0.819 (95% CI, 0.739–0.900). The sensitivity and specificity of phenylalanine were 75.5% and 81.4%, respectively, for detection of EGC. A multiple logistic regression analysis showed that high levels of aromatic amino acids in the gastric juice were associated with gastric cancer (adjusted β coefficients ranged from 1.801 to 4.414, P<0.001).
Increased levels of tyrosine, phenylalanine and tryptophan in the gastric juice samples were detected in the early phase of gastric carcinogenesis. Thus, tyrosine, phenylalanine and tryptophan in gastric juice could be used as biomarkers for the early detection of gastric cancer. A gastric juice analysis is an efficient, economical and convenient method for screening early gastric cancer development in the general population.
PMCID: PMC3496670  PMID: 23152906

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