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author:("Wang, honei")
1.  THE ANTI-MELANOMA ACTIVITY OF THE HISTONE DEACETYLASE INHIBITOR PANOBINOSTAT (LBH589) IS MEDIATED BY DIRECT TUMOR CYTOTOXICITY AND INCREASED TUMOR IMMUNOGENICITY 
Melanoma research  2013;10.1097/CMR.0b013e328364c0ed.
Objectives:
Melanoma is the deadliest skin cancer, and its incidence has been increasing faster than any other cancer. Although immunogenic, melanoma is not effectively cleared by host immunity. In this study, we investigate the therapeutic, anti-melanoma potential of the histone deacetylase inhibitor (HDACi) Panobinostat (LBH589) by assessing both its cytotoxic effects on melanoma cells as well as enhancement of immune recognition of melanoma.
Methods:
Utilizing murine and human melanoma cell lines, we analyze the effects of LBH589 on proliferation and survival. Additionally, we analyze expression of several immunologically relevant surface markers and melanoma differentiation antigens, and the ability of LBH589 treated melanoma to activate antigen specific T-cells. Finally, we assess the in vivo effects LBH589 in a mouse melanoma model.
Results:
Low nanomolar concentrations of LBH589 inhibit the growth of all melanoma cell lines tested, but not normal melanocytes. This inhibition is characterized by increased apoptosis as well as a G1 cell cycle arrest. In addition, LBH589 augments the expression of MHC and co-stimulatory molecules on melanoma cells leading to an increased ability to activate antigen specific T-cells. Treatment also increases expression of melanoma differentiation antigens. In vivo, LBH589 treatment of melanoma-bearing mice results in a significant increase in survival. However, in immunodeficient mice, the therapeutic effect of LBH589 is lost.
Conclusions:
Taken together, LBH589 exerts a dual effect upon melanoma cells by affecting not only growth/survival but also by increasing melanoma immunogenicity. These effects provide the framework for future evaluation of this HDAC inhibitor in melanoma treatment.
doi:10.1097/CMR.0b013e328364c0ed
PMCID: PMC4012016  PMID: 23963286
Melanoma; LBH589; Panobinostat; histone deacetylase inhibitors; HDAC
2.  Venous Thromboembolism Risk in Patients With Cancer Receiving Chemotherapy: A Real-World Analysis 
The Oncologist  2013;18(12):1321-1329.
The occurrence of malignant disease increases the risk for venous thromboembolism (VTE). This study sought to evaluate the risk for VTE in a large unselected cohort of patients with cancer receiving chemotherapy. Those undergoing chemotherapy as outpatients are at high risk for VTE and for major bleeding complications, and thromboprophylaxis should be considered for these patients.
Introduction.
The occurrence of malignant disease increases the risk for venous thromboembolism (VTE). Here we evaluate the risk for VTE in a large unselected cohort of patients with cancer receiving chemotherapy.
Methods.
The United States IMPACT health care claims database was retrospectively analyzed to identify patients with a range of solid tumors who started chemotherapy from January 2005 through December 2008. International Classification of Diseases, 9th revision, Clinical Modification Codes were used to identify cancer location, presence of VTE 3.5 months and 12 months after starting chemotherapy, and incidence of major bleeding complications. Health care costs were assessed one year before initiation of chemotherapy and one year after initiation of chemotherapy.
Results.
The overall incidence of VTE 3.5 months after starting chemotherapy was 7.3% (range 4.6%–11.6% across cancer locations) rising to 13.5% at 12 months (range 9.8%–21.3%). The highest VTE risk was identified in patients with pancreatic, stomach, and lung cancer. Patients in whom VTE developed had a higher risk for major bleeding at 3.5 months and at 12 months (11.0% and 19.8% vs. 3.8% and 9.6%, respectively). Health care costs were significantly higher in patients in whom VTE developed.
Conclusion.
Those undergoing chemotherapy as outpatients are at increased risk for VTE and for major bleeding complications. Thromboprophylaxis may be considered for such patients after carefully assessing the risks and benefits of treatment.
doi:10.1634/theoncologist.2013-0226
PMCID: PMC3868427  PMID: 24212499
Cancer; Chemotherapy; Health care costs; Thromboprophylaxis; Venous thromboembolism
3.  MDM4 Overexpressed in Acute Myeloid Leukemia Patients with Complex Karyotype and Wild-Type TP53 
PLoS ONE  2014;9(11):e113088.
Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10–15% of adult AML cases, and are often associated with a poor prognosis. Except for about 70% of CK-AML patients with biallelic inactivation of TP53, the leukemogenic mechanism in the nearly 30% of CK-AML patients with wild-type TP53 has remained elusive. In this study, 15 cases with complex karyotype and wild-type TP53 were screened out of 140 de novo AML patients and the expression levels of MDM4, a main negative regulator of p53-signaling pathway, were detected. We ruled out mutations in genes associated with a poor prognosis of CK-AML, including RUNX1 or FLT3-ITD. The mRNA expression levels of the full-length of MDM4 (MDM4FL) and short isoform MDM4 (MDM4S) were elevated in CK-AML relative to normal karyotype AML (NK-AML) patients. We also explored the impact of MDM4 overexpression on the cell cycle, cell proliferation and the spindle checkpoint of HepG2 cells, which is a human cancer cell line with normal MDM4 and TP53 expression. The mitotic index and the expression of p21, BubR1 and Securin were all reduced following Nocodazole treatment. Moreover, karyotype analysis showed that MDM4 overexpression might lead to aneuploidy or polyploidy. These results suggest that MDM4 overexpression is related to CK-AML with wild-type TP53 and might play a pathogenic role by inhibiting p53-signal pathway.
doi:10.1371/journal.pone.0113088
PMCID: PMC4236138  PMID: 25405759
4.  Mitophagy Enhances Oncolytic Measles Virus Replication by Mitigating DDX58/RIG-I-Like Receptor Signaling 
Journal of Virology  2014;88(9):5152-5164.
ABSTRACT
The success of future clinical trials with oncolytic viruses depends on the identification and the control of mechanisms that modulate their therapeutic efficacy. In particular, little is known about the role of autophagy in infection by attenuated measles virus of the Edmonston strain (MV-Edm). We investigated the interaction between autophagy, innate immune response, and oncolytic activity of MV-Edm, since the antiviral immune response is a known factor limiting virotherapies. We report that MV-Edm exploits selective autophagy to mitigate the innate immune response mediated by DDX58/RIG-I like receptors (RLRs) in non-small cell lung cancer (NSCLC) cells. Both RNA interference (RNAi) and overexpression approaches demonstrate that autophagy enhances viral replication and inhibits the production of type I interferons regulated by RLRs. We show that MV-Edm unexpectedly triggers SQSTM1/p62-mediated mitophagy, resulting in decreased mitochondrion-tethered mitochondrial antiviral signaling protein (MAVS) and subsequently weakening the innate immune response. These results unveil a novel infectious strategy based on the usurpation of mitophagy leading to mitigation of the innate immune response. This finding provides a rationale to modulate autophagy in oncolytic virotherapy.
IMPORTANCE In vitro studies, preclinical experiments in vivo, and clinical trials with humans all indicate that oncolytic viruses hold promise for cancer therapy. Measles virus of the Edmonston strain (MV-Edm), which is an attenuated virus derived from the common wild-type measles virus, is paradigmatic for therapeutic oncolytic viruses. MV-Edm replicates preferentially in and kills cancer cells. The efficiency of MV-Edm is limited by the immune response of the host against viruses. In our study, we revealed that MV-Edm usurps a homeostatic mechanism of intracellular degradation of mitochondria, coined mitophagy, to attenuate the innate immune response in cancer cells. This strategy might provide a replicative advantage for the virus against the development of antiviral immune responses by the host. These findings are important since they may not only indicate that inducers of autophagy could enhance the efficacy of oncolytic therapies but also provide clues for antiviral therapy by targeting SQSTM1/p62-mediated mitophagy.
doi:10.1128/JVI.03851-13
PMCID: PMC3993837  PMID: 24574393
5.  Reoperation after lumbar disc surgery in two hundred and seven patients 
International Orthopaedics  2013;37(8):1511-1517.
Purpose
The purpose of this study was to compare the causes and characteristics of reoperations after different primary operations for lumbar disc herniation (LDH).
Methods
Out of a series of 5,280 patients who underwent operations for LDH between 2001 and 2012, 207 patients (135 male and 72 female, mean age 47.7 years) underwent primary and revision operations, which were included in this study. The following clinical parameters were retrospectively assessed: the primary surgical methods, the intervals between primary and revision operations, and surgical findings in the revisions.
Results
In total, 232 lumbar discs underwent reoperations. One hundred and nineteen reoperations were performed after microendoscopic discectomy (MED group), 68 after percutaneous endoscopic lumbar discectomy (PELD group) and 45 after open disc surgery (open group). The locations of revision operations had priority over those of primary surgery, with a moderate correlation (kappa coefficient = 0.533). A total of 46.6 % of reoperations were performed within 0.5 years after primary surgery, and 35.3 % were performed between one and five years. Real recurrent herniation (homolateral herniations at the same level) was significantly more common than other reoperative surgical findings (70.6 % in PELD group, 47.1 % in MED group, 37.8 % in open group). The overall mean interval until revision surgery was 18.9 months (8.1 months in the PELD group vs. 19.7 months in the MED group vs. 33.1 months in the open group, p < 0.01).
Conclusions
For LDH, real recurrent herniation was the most common cause of reoperations, and more reoperations for real recurrent herniations and shorter intervals were found after minimally invasive endoscopic discectomy than after open disc surgery.
doi:10.1007/s00264-013-1925-2
PMCID: PMC3728382  PMID: 23695881
6.  Mitophagy promotes replication of oncolytic Newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells 
Oncotarget  2014;5(15):6365-6374.
Apoptosis contributes to antitumor effect of Newcastle disease virus (NDV). Autophagy is a protective response under cellular stress including viral infection. How autophagy interferes with oncolysis of NDV remains unclear. In this study, we found that NDV La Sota strain induced autophagy and preserved autophagic flux in non-small cell lung cancer cells. NDV-induced autophagy promoted viral replication by blocking cancer cells from caspase-dependent apoptosis. Moreover, we found that NDV recruited SQSTM1-mediated mitophagy to control cytochrome c release, and thus blocked intrinsic pro-apoptotic signaling. Finally, we observed an enhanced oncolysis in NSCLC cells treated with NDV in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Interestingly, a more profound antitumor effect could be achieved when administration of 3-MA was postponed to 24 h after NDV infection. Our findings unveil a novel way that NDV subverts mitophagy to favor its replication by blocking apoptosis, and provide rationale for systemic therapeutic cohort combining NDV with autophagy inhibitors in cancer therapy.
PMCID: PMC4171636  PMID: 25051374
Newcastle disease virus; mitophagy; apoptosis; autophagy; cancer
7.  Deconvolution of the Gene Expression Profiles of Valuable Banked Blood Specimens for Studying the Prognostic Values of Altered Peripheral Immune Cell Proportions in Cancer Patients 
PLoS ONE  2014;9(6):e100934.
Background
The altered composition of immune cells in peripheral blood has been reported to be associated with cancer patient survival. However, analysis of the composition of peripheral immune cells are often limited in retrospective survival studies employing banked blood specimens with long-term follow-up because the application of flow cytometry to such specimens is problematic. The aim of this study was to demonstrate the feasibility of deconvolving blood-based gene expression profiles (GEPs) to estimate the proportions of immune cells and determine their prognostic values for cancer patients.
Methods and Results
Here, using GEPs from peripheral blood mononuclear cells (PBMC) of 108 non-small cell lung cancer (NSCLC) patients, we deconvolved the immune cell proportions and analyzed their association with patient survival. Univariate Kaplan-Meier analysis showed that a low proportion of T cells was significantly associated with poor patient survival, as was the proportion of T helper cells; however, only the proportion of T cells was independently prognostic for patients by a multivariate Cox regression analysis (hazard ratio = 2.23; 95% CI, 1.01–4.92; p = .048). Considering that altered peripheral blood compositions can reflect altered immune responses within the tumor microenvironment, based on a tissue-based GEPs of NSCLC patients, we demonstrated a significant association between poor patient survival and the low level of antigen presentation, which play a critical role in T cell proliferation.
Conclusions
These results demonstrate that it is feasible to deconvolve GEPs from banked blood specimens for retrospective survival analysis of alterations of immune cell composition, and suggest the proportion of T cells in PBMC which might reflect the antigen presentation level within the tumor microenvironment can be a prognostic marker for NSCLC patients.
doi:10.1371/journal.pone.0100934
PMCID: PMC4069164  PMID: 24959668
8.  BCL11A gene DNA methylation contributes to the risk of type 2 diabetes in males 
BCL11A is a critical modulator involved in hemoglobin switching. Recent studies have established an association between BCL11A gene polymorphisms and a risk of type 2 diabetes (T2D). The aim of the present study was to assess the correlation between BCL11A DNA methylation and T2D. A total of 48 T2D cases and 48 age- and gender-matched controls were recruited to evaluate BCL11A methylation using bisulfite pyrosequencing technology. Although no significant association was observed in BCL11A methylation between T2D patients and healthy controls (P=0.322), breakdown analysis by gender identified a significant association between BCL11A methylation and T2D in males (P=0.018). Notably, there was also a significant female-specific association between the mean BCL11A DNA methylation and triglyceride (TG) concentration (r=−0.34; P=0.019). The results indicated that BCL11A methylation contributed to the risk of T2D in males. In addition, BCL11A methylation may have an effect on the development of T2D by influencing TG metabolism. Thus, gender difference may provide new information to aid the understanding of T2D pathogenesis.
doi:10.3892/etm.2014.1783
PMCID: PMC4079426  PMID: 25009601
type 2 diabetes; BCL11A; DNA methylation; CpG island; gender
9.  Treatment of Unstable Thoracolumbar Fractures through Short Segment Pedicle Screw Fixation Techniques Using Pedicle Fixation at the Level of the Fracture: A Finite Element Analysis 
PLoS ONE  2014;9(6):e99156.
Objective
To compare the von Mises stresses of the internal fixation devices among different short segment pedicle screw fixation techniques to treat thoracic 12 vertebral fractures, especially the mono-segment pedicle screw fixation and intermediate unilateral pedicle screw fixation techniques.
Methods
Finite element methods were utilised to investigate the biomechanical comparison of the four posterior short segment pedicle screw fixation techniques (S4+2: traditional short-segment 4 pedicle screw fixation [SPSF]; M4+2: mono-segment pedicle screw fixation; I6+2: intermediate bilateral pedicle screw fixation; and I5+2: intermediate unilateral pedicle screw fixation).
Results
The range of motion (ROM) in flexion, axial rotation, and lateral bending was the smallest in the I6+2 fixation model, followed by the I5+2 and S4+2 fixation models, but lateral bending was the largest in the M4+2 fixation model. The maximal stress of the upper pedicle screw is larger than the lower pedicle screw in S4+2 and M4+2. The largest maximal von Mises stress was observed in the upper pedicle screw in the S4+2 and M4+2 fixation models and in the lower pedicle screw in the I6+2 and I5+2 fixation models. The values of the largest maximal von Mises stress of the pedicle screws and rods during all states of motion were 263.1 MPa and 304.5 MPa in the S4+2 fixation model, 291.6 MPa and 340.5 MPa in the M4+2 fixation model, 182.9 MPa and 263.2 MPa in the I6+2 fixation model, and 269.3 MPa and 383.7 MPa in the I5+2 fixation model, respectively. Comparing the stress between different spinal loadings, the maximal von Mises stress of the implants were observed in flexion in all implanted models.
Conclusion
Additional bilateral pedicle screws at the level of the fracture to SPSF may result in a stiffer construct and less von Mises stress for pedicle screws and rods. The largest maximal von Mises stress of the pedicle screws during all states of motion were observed in the mono-segment pedicle screw fixation technique.
doi:10.1371/journal.pone.0099156
PMCID: PMC4051693  PMID: 24914815
10.  Current and Emerging Biomarkers of Cell Death in Human Disease 
BioMed Research International  2014;2014:690103.
Cell death is a critical biological process, serving many important functions within multicellular organisms. Aberrations in cell death can contribute to the pathology of human diseases. Significant progress made in the research area enormously speeds up our understanding of the biochemical and molecular mechanisms of cell death. According to the distinct morphological and biochemical characteristics, cell death can be triggered by extrinsic or intrinsic apoptosis, regulated necrosis, autophagic cell death, and mitotic catastrophe. Nevertheless, the realization that all of these efforts seek to pursue an effective treatment and cure for the disease has spurred a significant interest in the development of promising biomarkers of cell death to early diagnose disease and accurately predict disease progression and outcome. In this review, we summarize recent knowledge about cell death, survey current and emerging biomarkers of cell death, and discuss the relationship with human diseases.
doi:10.1155/2014/690103
PMCID: PMC4052120  PMID: 24949464
11.  Expression of Serum Exosomal MicroRNA-21 in Human Hepatocellular Carcinoma 
BioMed Research International  2014;2014:864894.
New strategies for the diagnosis of hepatocellular carcinoma (HCC) are urgently needed. There is an increasing interest in using microRNAs (miRNAs) as biomarkers in diseases. In this study, we examined the expression of miR-21 in serum exosomes from patients with HCC or chronic hepatitis B (CHB) and investigated the potential clinical significance of miR-21. Quantitative RT-PCR indicated that the concentration of miR-21 was significantly higher in exosomes than in exosome-depleted supernatants or the whole serum. Further, the expression level of serum exosomal miR-21 was significantly higher in patients with HCC than those with CHB or healthy volunteers (P < 0.0001, P < 0.0001, resp.). High level of miR-21 expression correlated with cirrhosis (P = 0.024) and advanced tumor stage (P = 0.001). Although serum level of miR-21 was higher in patients with HCC than in patients with CHB and healthy volunteers, the sensitivity of detection is much lower than using exosomal miR-21. These findings indicate that miR-21 is enriched in serum exosomes which provides increased sensitivity of detection than whole serum. Exosomal miR-21 may serve as a potential biomarker for HCC diagnosis.
doi:10.1155/2014/864894
PMCID: PMC4052145  PMID: 24963487
12.  HISTONE DEACETYLASE INHIBITOR LAQ824 AUGMENTS INFLAMMATORY RESPONSES IN MACROPHAGES THROUGH TRANSCRIPTIONAL REGULATION OF IL-10 
Antigen-presenting cells (APCs) are important in the initiation of productive antigen-specific T-cell responses and in the induction of T-cell anergy. The inflammatory status of the APC at the time of encounter with antigen specific T-cells plays a central role in determining such divergent T-cell outcomes. A better understanding of the regulation of pro-inflammatory and anti-inflammatory genes in its natural setting, the chromatin substrate, might provide novel insights to overcome anergic mechanisms mediated by APCs. Here we show for the first time that treatment of BALB/c murine macrophages with the histone deacetylase inhibitor (HDI) LAQ824, induces chromatin changes at the level of the IL-10 gene promoter that leads to enhanced recruitment of the transcriptional repressors HDAC11 and PU.1. Such an effect is associated with diminished IL-10 production and induction of inflammatory cells able of priming naïve antigen-specific T-cells, but more importantly, capable of restoring the responsiveness of anergized antigen-specific CD4+ T-cells.
doi:10.4049/jimmunol.1001101
PMCID: PMC3998678  PMID: 21368229
13.  STAT3 INHIBITION AUGMENTS THE IMMUNOGENICITY OF MALIGNANT B-CELLS LEADING TO EFFECTIVE ANTITUMOR IMMUNITY IN B-CELL LYMPHOMAS 
Cancer research  2012;72(17):4440-4448.
Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell Non-Hodgkin’s lymphomas characterized by an initial response to first-line treatment with chemotherapy plus monoclonal antibodies followed by relapse and less responsiveness to further lines of treatment. Harnessing the immune system to elicit its exquisite specificity and long-lasting protection might provide sustained MCL immunity that could potentially eradicate residual malignant cells responsible for disease relapse. Here we show that genetic or pharmacologic disruption of Stat3 in malignant B-cells augments their immunogenicity leading to better activation of antigen-specific CD4+ T-cells and restoration of responsiveness of tolerized T-cells. The additional demonstration that in vivo treatment of MCL-bearing mice with a specific Stat3 inhibitor resulted in decreased Stat3 phosphorylation in malignant B-cells and anti-lymphoma immunity, points to Stat3 inhibition as an enticing strategy to overcome tolerance to tumor antigens and elicit a strong immunity against MCL and other B-cell malignancies.
doi:10.1158/0008-5472.CAN-11-3619
PMCID: PMC3990429  PMID: 22728650
Stat3; anti-tumor immunity; B-cell lymphoma
14.  HPV E6/E7 RNA In Situ Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade 
PLoS ONE  2014;9(3):e91142.
Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.
doi:10.1371/journal.pone.0091142
PMCID: PMC3953338  PMID: 24625757
15.  RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma 
The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.
doi:10.3791/51426
PMCID: PMC4145807  PMID: 24637627
Medicine; Issue 85; RNAscope; Head and Neck Squamous Cell Carcinoma (HNSCC); Oropharyngeal Squamous Cell Carcinoma (OPSCC); Human Papillomavirus (HPV); E6/ E7 mRNA; in situ hybridization; tumor
16.  A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis 
Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method (in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined).
doi:10.1002/ijc.27739
PMCID: PMC3498587  PMID: 22821242
human papillomavirus; oropharyngeal cancer; prognosis; expression signature
17.  Papillary Squamous Cell Carcinoma of the Head and Neck 
A relationship between human papillomavirus (HPV) infection and papillary squamous cell carcinoma (PSCC) has been suggested. However, to date, no studies have thoroughly and directly evaluated for transcriptional activity of the virus or the clinicopathologic significance of HPV-positive PSCC. Forty-eight cases of PSCC were retrieved from our surgical pathology database and were reviewed by 4 study pathologists, with tumors defined as SCC with a significant component of papillary growth in the tumor. Immunohistochemical analysis for p16 and p53 was performed. Overexpression of p16 was used as a surrogate marker of transcriptionally active HPV. Transcriptional activity was also directly evaluated using RNA in situ hybridization to detect high-risk HPV E6/E7 mRNA. Clinical follow-up data were obtained by chart review. Seven cases were located in the oral cavity, 19 in the oropharynx, and 22 in the larynx. Two morphologic types of PSCC were identified: keratinizing type, in which the epithelial cells showed a maturation trend with minimal surface parakeratin, and nonkeratinizing type, in which the papillae were completely covered by immature basaloid cells. Transcriptionally active HPV was present in 23 of 43 (53.4%) tumors. The majority of tumors harboring transcriptionally active HPV arose in the oropharynx, showed nonkeratinizing morphology, were p16 positive, and p53 negative. Transcriptionally active HPV was also present in many laryngeal and oral cavity PSCCs. Overall survival, disease-specific survival, and disease-free survival were favorable and did not significantly differ by anatomic subsite. However, HPV-related tumors showed a trend toward better survival.
doi:10.1097/PAS.0b013e318290427d
PMCID: PMC3903508  PMID: 23797720
papillary squamous cell carcinoma; head and neck; human papillomavirus; p16; RNA in situ hybridization
18.  A Test of Rensch’s Rule in Greater Horseshoe Bat (Rhinolophus ferrumequinum) with Female-Biased Sexual Size Dimorphism 
PLoS ONE  2014;9(1):e86085.
Sexual size dimorphism (SSD) is widespread within the animal kingdom. Rensch’s rule describes a relationship between SSD and body size: SSD increases with body size when males are the larger sex, and decreases with body size when females are the larger sex. Rensch’s rule is well supported for taxa that exhibit male-biased SSD but patterns of allometry among taxa with female-biased size dimorphism are mixed, there is evidence both for and against the rule. Furthermore, most studies have investigated Rensch’s rule across a variety of taxa; but among-population studies supporting Rensch’s rule are lacking, especially in taxa that display only slight SSD. Here, we tested whether patterns of intraspecific variation in SSD in greater horseshoe bats conform to Rensch’s rule, and evaluated the contribution of latitude to Rensch’s rule. Our results showed SSD was consistently female-biased in greater horseshoe bats, although female body size was only slightly larger than male body size. The slope of major axis regression of log10 (male) on log10 (female) was significantly different from 1. Forearm length for both sexes of greater horseshoe bats was significantly negatively correlated with latitude, and males displayed a slightly but nonsignificant steeper latitudinal cline in body size than females. We suggest that variation in patterns of SSD among greater horseshoe bat populations is consistent with Rensch’s rule indicating that males were the more variable sex. Males did not have a steeper body size–latitude relationship than females suggesting that sex-specific latitudinal variation in body size may not be an important contributing factor to Rensch’s rule. Future research on greater horseshoe bats might best focus on more comprehensive mechanisms driving the pattern of female-biased SSD variation.
doi:10.1371/journal.pone.0086085
PMCID: PMC3896443  PMID: 24465886
19.  A Novel PCR-Based Approach to Discover miRNA Target Genes 
MiRNAs are potent regulators of gene expression, and most miRNAs have from several to several thousands of gene targets. Validating the numerous gene targets of a given miRNA remains challenging despite the existence of various tools and databases that predict candidate gene-miRNA pairs. In the present study, we present a high-throughput but flexible method that applies a PCR-based application to simulate the binding of miRNAs to their gene targets. Using hsa-miR-377 as an illustrative example, our method was able to identify 13 potential targets of hsa-miR-377. Moreover, our results include 2 genes (SOD2 and PPM1A) that have already been verified as targets of hsa-miR-377. Our method may provide an alternative way of identifying the gene targets of miRNAs for future research.
doi:10.7150/ijms.9343
PMCID: PMC4196129  PMID: 25317074
miRNA; gene target; hsa-miR-377; high-throughput.
20.  Grateloupia tenuis Wang et Luan sp. nov. (Halymeniaceae, Rhodophyta): A New Species from South China Sea Based on Morphological Observation and rbcL Gene Sequences Analysis 
BioMed Research International  2013;2013:560163.
Grateloupia tenuis Wang et Luan sp. nov. is a new species described from Lingshui, Hainan Province, South China Sea. Based on the external form and internal structure, combined with rbcL gene sequence analysis, Grateloupia tenuis is distinct from other Grateloupia species as follows: (1) thalli is slippery and cartilaginous in texture; possess fewer branches, relatively slight main axes, and two or three dichotomous branches; (2) cortex is 5-6 layers; medulla is solid when young, but hollow in old branches; reproductive structures are dispersed in main axes of thalli and lower portions of branchlets; exhibits Grateloupia-type auxiliary cell ampullae; (3) the four studied G. tenuis sequences were positioned in a large Grateloupia clade of Halymeniaceae, which included sister group generitype G. filicina with 68 bp differences; G. tenuis was determined to be a sister taxon to the G. catenata, G. ramosissima, G. orientalis, and G. filiformis subclade. The pairwise distances between G. tenuis and these species were 39 to 50 bp. The sequences of G. tenuis differed by 81–108 bp from the sequences of other samples in Grateloupia; there are 114–133 bp changes between G. tenuis and other genera of Halymeniaceae. In final analysis, we considered Grateloupia tenuis Wang et Luan sp. nov. to be a new species of genus Grateloupia.
doi:10.1155/2013/560163
PMCID: PMC3885149  PMID: 24455703
21.  Tumor Evolution and Intratumor Heterogeneity of an Oropharyngeal Squamous Cell Carcinoma Revealed by Whole-Genome Sequencing12 
Neoplasia (New York, N.Y.)  2013;15(12):1371-1378.
Head and neck squamous cell carcinoma (HNSCC) is characterized by significant genomic instability that could lead to clonal diversity. Intratumor clonal heterogeneity has been proposed as a major attribute underlying tumor evolution, progression, and resistance to chemotherapy and radiation. Understanding genetic heterogeneity could lead to treatments specific to resistant and metastatic tumor cells. To characterize the degree of intratumor genetic heterogeneity within a single tumor, we performed whole-genome sequencing on three separate regions of an human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma and two separate regions from one corresponding cervical lymph node metastasis. This approach achieved coverage of approximately 97.9% of the genome across all samples. In total, 5701 somatic point mutations (SPMs) and 4347 small somatic insertions and deletions (indels)were detected in at least one sample. Ninety-two percent of SPMs and 77% of indels were validated in a second set of samples adjacent to the discovery set. All five tumor samples shared 41% of SPMs, 57% of the 1805 genes with SPMs, and 34 of 55 cancer genes. The distribution of SPMs allowed phylogenetic reconstruction of this tumor's evolutionary pathway and showed that the metastatic samples arose as a late event. The degree of intratumor heterogeneity showed that a single biopsy may not represent the entire mutational landscape of HNSCC tumors. This approach may be used to further characterize intratumor heterogeneity in more patients, and their sample-to-sample variations could reveal the evolutionary process of cancer cells, facilitate our understanding of tumorigenesis, and enable the development of novel targeted therapies.
PMCID: PMC3884528  PMID: 24403859
22.  Astragaloside IV Attenuates Experimental Autoimmune Encephalomyelitis of Mice by Counteracting Oxidative Stress at Multiple Levels 
PLoS ONE  2013;8(10):e76495.
Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease found mostly in young adults in the western world. Oxidative stress induced neuronal apoptosis plays an important role in the pathogenesis of MS. In current study, astragaloside IV (ASI), a natural saponin molecule isolated from Astragalus membranceus, given at 20 mg/kg daily attenuated the severity of experimental autoimmune encephalomyelitis (EAE) in mice significantly. Further studies disclosed that ASI treatment inhibited the increase of ROS and pro-inflammatory cytokine levels, down-regulation of SOD and GSH-Px activities, and elevation of iNOS, p53 and phosphorylated tau in central nervous system (CNS) as well as the leakage of BBB of EAE mice. Meanwhile, the decreased ratio of Bcl-2/Bax was reversed by ASI. Moreover, ASI regulated T-cell differentiation and infiltration into CNS. In neuroblast SH-SY5Y cells, ASI dose-dependently reduced cellular ROS level and phosphorylation of tau in response to hydrogen peroxide challenge by modulation of Bcl-2/Bax ratio. ASI also inhibited activation of microglia both in vivo and in vitro. iNOS up-regulation induced by IFNγ stimulation was abolished by ASI dose-dependently in BV-2 cells. In summary, ASI prevented the severity of EAE progression possibly by counterbalancing oxidative stress and its effects via reduction of cellular ROS level, enhancement of antioxidant defense system, increase of anti-apoptotic and anti-inflammatory pathways, as well as modulation of T-cell differentiation and infiltration into CNS. The study suggested ASI may be effective for clinical therapy/prevention of MS.
doi:10.1371/journal.pone.0076495
PMCID: PMC3790693  PMID: 24124567
23.  Genome-Wide Analysis of ZmDREB Genes and Their Association with Natural Variation in Drought Tolerance at Seedling Stage of Zea mays L 
PLoS Genetics  2013;9(9):e1003790.
The worldwide production of maize (Zea mays L.) is frequently impacted by water scarcity and as a result, increased drought tolerance is a priority target in maize breeding programs. While DREB transcription factors have been demonstrated to play a central role in desiccation tolerance, whether or not natural sequence variations in these genes are associated with the phenotypic variability of this trait is largely unknown. In the present study, eighteen ZmDREB genes present in the maize B73 genome were cloned and systematically analyzed to determine their phylogenetic relationship, synteny with rice, maize and sorghum genomes; pattern of drought-responsive gene expression, and protein transactivation activity. Importantly, the association between the nucleic acid variation of each ZmDREB gene with drought tolerance was evaluated using a diverse population of maize consisting of 368 varieties from tropical and temperate regions. A significant association between the genetic variation of ZmDREB2.7 and drought tolerance at seedling stage was identified. Further analysis found that the DNA polymorphisms in the promoter region of ZmDREB2.7, but not the protein coding region itself, was associated with different levels of drought tolerance among maize varieties, likely due to distinct patterns of gene expression in response to drought stress. In vitro, protein-DNA binding assay demonstrated that ZmDREB2.7 protein could specifically interact with the target DNA sequences. The transgenic Arabidopsis overexpressing ZmDREB2.7 displayed enhanced tolerance to drought stress. Moreover, a favorable allele of ZmDREB2.7, identified in the drought-tolerant maize varieties, was effective in imparting plant tolerance to drought stress. Based upon these findings, we conclude that natural variation in the promoter of ZmDREB2.7 contributes to maize drought tolerance, and that the gene and its favorable allele may be an important genetic resource for the genetic improvement of drought tolerance in maize.
Author Summary
Water scarcity is one of the most severe threats to maize production worldwide. Although research has demonstrated that DREB-type transcription factors play important roles in plant water stress response, whether the specific genetic variants in DREB genes contribute to plant drought tolerance is largely unknown. Taking advantages of recent technical and methodological advance, we systematically analyzed all the functional DREB genes in maize and examined their associations with the natural variation in drought tolerance of 368 maize varieties collected from tropical and temperate regions. A significant association in the ZmDREB2.7 gene with drought tolerance was detected in that the DNA polymorphisms in the gene promoter region, but not those in the protein coding region, contributed to observed variations in maize drought tolerance, probably due to the distinct gene expression patterns in response to the stress. Overexpressing ZmDREB2.7 in Arabidopsis resulted in enhanced tolerance to drought stress. Moreover, a favorable ZmDREB2.7 allele, identified from drought-tolerant varieties, was effective in improving plant tolerance to drought stress when it was introduced into a drought-sensitive background. ZmDREB2.7 and its favorable allele represent a valuable genetic resource for enhancing maize drought tolerance by marker assisted breeding and transformation technology.
doi:10.1371/journal.pgen.1003790
PMCID: PMC3784558  PMID: 24086146
24.  A peptide-mediated targeting gene delivery system for malignant glioma cells 
Glioblastoma multiforme (GBM) is the most common and malignant glioma. Although there has been considerable progress in treatment strategies, the prognosis of many patients with GBM remains poor. In this work, polyethylenimine (PEI) and the VTWTPQAWFQWV (VTW) peptide were modified and synthesized into GBM-targeting nanoparticles. The transfection efficiency of U-87 (human glioblastoma) cells was evaluated using fluorescence microscopy and flow cytometry. Cell internalization was investigated to verify the nanoparticle delivery into the cytoplasm. Results showed that the methods of polymer conjugation and the amount of VTW peptide were important factors to polymer synthesis and transfection. The PEI-VTW20 nanoparticles increased the transfection efficiency significantly. This report describes the use of VTW peptide-based PEI nanoparticles for intracellular gene delivery in a GBM cell-specific manner.
doi:10.2147/IJN.S44990
PMCID: PMC3790891  PMID: 24101872
glioblastoma; polyethylenimine; nanoparticles; drug-delivery systems; gene transfer techniques
25.  Correction: Epigenetic Regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA Methylation and Histone Modification 
PLoS ONE  2013;8(9):10.1371/annotation/1b1eb36e-60a4-4d28-bb3a-ee5a12a3d5d8.
doi:10.1371/annotation/1b1eb36e-60a4-4d28-bb3a-ee5a12a3d5d8
PMCID: PMC3779267

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