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1.  Optimal Allocation of Gold Standard Testing under Constrained Availability: Application to Assessment of HIV Treatment Failure 
The World Health Organization (WHO) guidelines for monitoring the effectiveness of HIV treatment in resource-limited settings (RLS) are mostly based on clinical and immunological markers (e.g., CD4 cell counts). Recent research indicates that the guidelines are inadequate and can result in high error rates. Viral load (VL) is considered the “gold standard”, yet its widespread use is limited by cost and infrastructure. In this paper, we propose a diagnostic algorithm that uses information from routinely-collected clinical and immunological markers to guide a selective use of VL testing for diagnosing HIV treatment failure, under the assumption that VL testing is available only at a certain portion of patient visits. Our algorithm identifies the patient sub-population, such that the use of limited VL testing on them minimizes a pre-defined risk (e.g., misdiagnosis error rate). Diagnostic properties of our proposal algorithm are assessed by simulations. For illustration, data from the Miriam Hospital Immunology Clinic (RI, USA) are analyzed.
PMCID: PMC3963362  PMID: 24672142
Antiretroviral failure; constrained optimization; HIV/AIDS; resource limited; ROC; tripartite classification
2.  Berberine represses DAXX gene transcription and induces cancer cell apoptosis 
DAXX is a multifunctional protein that regulates a wide range of cellular signaling pathways for both cell survival and apoptosis. Regulation of DAXX gene expression remains largely obscure. We recently reported that berberine (BBR), a natural product derived from a plant used in Chinese herbal medicine, downregulates DAXX expression at the transcriptional level. Here, we further investigate the mechanisms underlying the transcriptional suppression of DAXX by BBR. By analyzing and mapping the putative DAXX gene promoter, we identified the core promoter region (from −161 to −1), which contains consensus sequences for the transcriptional factors Sp1 and Ets1. We confirmed that Sp1 and Ets1 bound to the core promoter region of DAXX and stimulated DAXX transcriptional activity. In contrast, BBR bound to the DAXX core promoter region and suppressed its transcriptional activity. Following studies demonstrated a possible mechanism that BBR inhibited the DAXX promoter activity through blocking or disrupting the association of Sp1 or Ets1 and their consensus sequences in the promoter. Downregulation of DAXX by BBR resulted in inhibition of MDM2 and subsequently, activation of p53, leading to cancer cell death. Our results reveal a novel possible mechanism: by competitively binding to the Sp1 and Ets1 consensus sequences, BBR inhibits the transcription of DAXX, thus inducing cancer cell apoptosis through a p53-dependent pathway.
PMCID: PMC3961588  PMID: 23295648
DAXX; Sp1; Ets1; BBR; neuroblastoma
3.  Discovery of a Strongly-Interrelated Gene Network in Corals under Constant Darkness by Correlation Analysis after Wavelet Transform on Complex Network Model 
PLoS ONE  2014;9(3):e92434.
Coral reefs occupy a relatively small portion of sea area, yet serve as a crucial source of biodiversity by establishing harmonious ecosystems with marine plants and animals. Previous researches mainly focused on screening several key genes induced by stress. Here we proposed a novel method—correlation analysis after wavelet transform of complex network model, to explore the effect of light on gene expression in the coral Acropora millepora based on microarray data. In this method, wavelet transform and the conception of complex network were adopted, and 50 key genes with large differences were finally captured, including both annotated genes and novel genes without accurate annotation. These results shed light on our understanding of coral's response toward light changes and the genome-wide interaction among genes under the control of biorhythm, and hence help us to better protect the coral reef ecosystems. Further studies are needed to explore how functional connections are related to structural connections, and how connectivity arises from the interactions within and between different systems. The method introduced in this study for analyzing microarray data will allow researchers to explore genome-wide interaction network with their own dataset and understand the relevant biological processes.
PMCID: PMC3961355  PMID: 24651851
4.  Systematic Review and Meta-Analysis of the Use of Phosphodiesterase Type 5 Inhibitors for Treatment of Erectile Dysfunction following Bilateral Nerve-Sparing Radical Prostatectomy 
PLoS ONE  2014;9(3):e91327.
Prostate cancer is relatively common cancer occurring in males. Radical prostatectomy (RP) is the most effective treatment for a localized tumor but erectile dysfunction (ED) is common complication, even when bilateral nerve-sparing RP (BNSRP) is performed. Clinical trials have shown varied effectiveness of phosphodiesterase type-5 inhibitors (PDE5-Is) for treatment of post-BNSRP ED, but there remains controversy over the application of this treatment and no formal systematic review and meta-analysis for the use of PDE5-Is for this condition has been conducted. This review was to systematically assess the efficacy and safety of oral PDE5-Is for post-BNSRP ED. A database search was conducted to identify randomized controlled trials (RCTs). The comparative efficacy of treatments was analyzed by fixed or random effect modeling. Erectile function was measured using the International Index of Erectile Function (IIEF), Sexual Encounter Profile (SEP) question-2, 3 and the Global Assessment Question (GAQ). The rate and incidence of adverse events (AEs) were determined. The quality of included studies was appraised using the Cochrane Collaboration bias appraisal tool. Eight RCTs were included in the analyses. PDE5-Is were effective for treating post-BNSRP ED compared to placebo when erectile function was determined using the IIEF score [mean difference (MD) 5.63, 95% confidence interval (CI) (4.26–6.99)], SEP-2 [relative risk (RR) 1.63, 95% CI (1.18–2.25) ], SEP-3 [RR 2.00, 95% CI (1.27–3.15) ] and GAQ [RR 3.35, 95% CI (2.68–4.67) ]. The subgroup analysis could find a trend that longer treatment duration, higher dosage, on-demand dosing, sildenafil and mild ED are associated with more responsiveness to PDE5-Is. PDE5-Is were overall well tolerated with headache being the most commonly reported AE. Our data provides compelling evidence for the use of PDE5-Is as a primary treatment for post-BNSRP ED. However, further studies are required to optomize usage parameters (such as dosage and duration of treatment).
PMCID: PMC3949994  PMID: 24618671
5.  High Expression of Neuro-Oncological Ventral Antigen 1 Correlates with Poor Prognosis in Hepatocellular Carcinoma 
PLoS ONE  2014;9(3):e90955.
Neuro-oncological ventral antigen 1 (Nova1) is a neuron-specific RNA-binding protein in human paraneoplastic opsoclonus-myoclonus ataxia accompanying with malignant tumors, but its role in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that overexpressed intratumoral Nova1 was associated with poor survival rate and increased recurrence rate of HCC, especially early recurrence, and was an independent prognostic factor for overall survival rate and tumor recurrence. HCC cell lines over-expressing Nova1 exhibited greater potentials in cell proliferation, invasion and migration, while knockdown of Nova1 had the opposite effects. All these findings indicate that Nova1 may act as a prognostic marker for poor outcome and high recurrence in HCC.
PMCID: PMC3946567  PMID: 24608171
6.  Germline Transmission of an Embryonic Stem Cell Line Derived from BALB/c Cataract Mice 
PLoS ONE  2014;9(3):e90707.
Mice embryonic stem (ES) cells have enabled the generation of mouse strains with defined mutation(s) in their genome for putative disease loci analysis. In the study of cataract, the complex genetic background of this disease and lack of long-term self-renewal ES cells have hampered the functional researches of cataract-related genes. In this study, we aimed to establish ES cells from inherited cataract mice (BALB/CCat/Cat). Embryos of cataract mice were cultured in chemical-defined N2B27 medium with the presence of two small molecules PD0325901 and CHIR99021 (2i) and an ES cell line (named EH-BES) was successfully established. EH-BES showed long-term self-renewal in 2i medium and maintained capacity of germline transmission. Most importantly, the produced chimera and offspring developed congenital cataract as well. Flow cytometry assay revealed that EH-BES are homogeneous in expression of Oct4 and Rex1in 2i medium, which may account for their self-renewal ability. With long-term self-renewal ability and germline-competent, EH-BES cell line can facilitate genetic and functional researches of cataract-related genes and better address mechanisms of cataract.
PMCID: PMC3942454  PMID: 24595217
7.  Dynamic relations between fast-food restaurant and body weight status: a longitudinal and multilevel analysis of Chinese adults 
Mixed findings have been reported on the association between Western fast-food restaurants and body weight status. Results vary across study contexts and are sensitive to the samples, measures and methods used. Most studies have failed to examine the temporally dynamic associations between community exposure to fast-food restaurants and weight changes.
Bayesian hierarchical regressions are used to model changes in body mass index, waist-to-height ratio (WHtR) and waist-to-hip ratio (WHpR) as a function of changes in Western fast-food restaurants in 216 communities for more than 9000 Chinese adults followed up multiple times between 2000 and 2009.
Number of Western fast-food restaurants is positively associated with subsequent increases in WHtR and WHpR among rural population. More fast-food restaurants are positively associated with a future increase in WHpR for urban women. Increased availability of fast food between two waves is related to increased WHtR for urban men over the same period. A past increase in number of fast-food restaurants is associated with subsequent increases in WHtR and WHpR for rural population.
The associations between community exposure to Western fast food and weight changes are temporally dynamic rather than static. Improved measures of exposure to community environment are needed to achieve more precise estimates and better understanding of these relationships. In light of the findings in this study and China’s rapid economic growth, further investigation and increased public health monitoring is warranted since Western fast food is likely to be more accessible and affordable in the near future.
PMCID: PMC3574174  PMID: 22923769
8.  Comprehensive epigenetic analysis of Notch pathway in high-grade serous ovarian cancer 
Gynecologic oncology  2012;128(3):506-511.
Gene methylation and other epigenetic modifications of gene regulation have been implicated in the growth of ovarian cancer, but the clinical significance of such modifications in the Notch pathway in high-grade serous ovarian cancer (HGS-OvCa) is not well understood. We used The Cancer Genome Atlas (TCGA) data to study the clinical relevance of epigenetic modifications of Notch pathway genes.
We analyzed the interaction of DNA methylation and miRNAs with gene expression data for Notch family members with the Spearman rank correlation test and explored potential relationships with overall survival (OS) with the log-rank test. We downloaded clinical data, level 3 gene expression data, and level 3 DNA methylation data for 480 patients with stage II-IV HGS-OvCa from TCGA data portal. Patients were randomly divided into training and validation cohorts for survival analyses. In each set, patients were grouped into percentiles according to methylation and microRNA (miRNA) or messenger RNA (mRNA) levels. We used several algorithms to predict miRNA-mRNA interaction.
There were significant inverse relationships between methylation status and mRNA expression for PPARG, CCND1, and RUNX1. For each of these genes, patients with a lower methylation level and higher expression level had significantly poorer OS than did patients with a higher methylation level and lower expression level. We also found a significant inverse relationship between miRNAs and mRNA expression for CCND1, PPARG, and RUNX1. By further analyzing the effect of miRNAs on gene expression and OS, we found that patients with higher levels of CCND1, PPARG, and RUNX1 expression and lower expression levels of their respective miRNAs (502-5p, 128, and 215/625) had significantly poorer OS.
Epigenetic alterations of multiple Notch target genes and pathway interacting genes (PPARG, CCND1, and RUNX1) may relate to activation of this pathway and poor survival of patients with HGS-OvCa.
PMCID: PMC3645276  PMID: 23200915
Notch pathway; high-grade serous ovarian carcinoma; epigenetic alterations; High-grade serous ovarian cancer; The Cancer Genome Atlas; Epigenetic modifications of gene regulation
9.  A Suppressor of Cytokine Signaling 1 Antagonist Enhances Antigen-Presenting Capacity and Tumor Cell Antigen-Specific Cytotoxic T Lymphocyte Responses by Human Monocyte-Derived Dendritic Cells 
The suppressor of cytokine signaling 1 (SOCS1) has emerged as a critical inhibitory molecule for controlling the cytokine response and antigen presentation by dendritic cells (DCs), thereby regulating the magnitude of both innate and adaptive immunity. The aim of this study was to investigate whether the SOCS1 antagonist pJAK2(1001-1013) peptide can weaken or block the inhibition function of SOCS1 in DCs by evaluating the phenotype and cytokine production, antigen-presenting, and specific T-cell-activating capacities of DCs electroporated with human gastric cancer cell total RNA. Furthermore, STAT1 activation of the JAK/STAT signal pathway mediated by SOCS1 was analyzed by Western blotting. The results demonstrate that the SOCS1 antagonist pJAK2(1001-1013) peptide upregulated the expression of the maturation marker (CD83) and costimulatory molecule (CD86) of RNA-electroporated human monocyte-derived mature DCs (mDCs), potentiated the capacity of mDCs to induce T-cell proliferation, stimulated the secretion of proinflammatory cytokines, and enhanced the cytotoxicity of tumor cell antigen-specific CTLs activated by human gastric cancer cell total RNA-electroporated mDCs. Data from Western blot analysis indicate that STAT1 was further activated in pJAK2(1001-1013) peptide-loaded mDCs. These results imply that the SOCS1 antagonist pJAK2(1001-1013) peptide is an effective reagent for the enhancement of antigen-specific antitumor immunity by DCs.
PMCID: PMC3889590  PMID: 23885028
10.  Chloride intracellular channel 1 regulates colon cancer cell migration and invasion through ROS/ERK pathway 
AIM: To investigate the mechanisms of chloride intracellular channel 1 (CLIC1) in the metastasis of colon cancer under hypoxia-reoxygenation (H-R) conditions.
METHODS: Fluorescent probes were used to detect reactive oxygen species (ROS) in LOVO cells. Wound healing assay and transwell assay were performed to examine the migration and invasion of LOVO cells. Expression of CLIC1 mRNA and protein, p-ERK, MMP-2 and MMP-9 proteins was analyzed by reverse transcription-polymerase chain reaction and Western blot.
METHODS: H-R treatment increased the intracellular ROS level in LOVO cells. The mRNA and protein expression of CLIC1 was elevated under H-R conditions. Functional inhibition of CLIC1 markedly decreased the H-R-enhanced ROS generation, cell migration, invasion and phosphorylation of ERK in treated LOVO cells. Additionally, the expression of MMP-2 and MMP-9 could be regulated by CLIC1-mediated ROS/ERK pathway.
CONCLUSION: Our results suggest that CLIC1 protein is involved in the metastasis of colon cancer LOVO cells via regulating the ROS/ERK pathway in the H-R process.
PMCID: PMC3934477  PMID: 24587680
Colon cancer; Intracellular chloride channel 1; Hypoxia-reoxygenation; Reactive oxygen species; Extracellular signal-regulated kinase; Cancer invasion
11.  DNA Damage Induces Down-Regulation of Prp19 via Impairing Prp19 Stability in Hepatocellular Carcinoma Cells 
PLoS ONE  2014;9(2):e89976.
Pre-mRNA processing factor 19 (Prp19) activates pre-mRNA spliceosome and also mediates DNA damage response. Prp19 overexpression in cells with functional p53 leads to decreased apoptosis and increases cell survival after DNA damage. Here we showed that in hepatocellular carcinoma (HCC) cells with inactive p53 or functional p53, Prp19 was down-regulated due to the impaired stability under chemotherapeutic drug treatment. Silencing Prp19 expression enhanced apoptosis of HCC cells with or without chemotherapeutic drug treatment. Furthermore high level of Prp19 may inhibit chemotherapeutic drugs induced apoptosis in hepatocellular carcinoma cells through modulating myeloid leukemia cell differentiation 1 expression. These results indicated that targeting Prp19 may potentiate pro-apoptotic effect of chemotherapeutic agents on HCC.
PMCID: PMC3938560  PMID: 24587161
12.  Silencing Alpha-Fetoprotein Inhibits VEGF and MMP-2/9 Production in Human Hepatocellular Carcinoma Cell 
PLoS ONE  2014;9(2):e90660.
Alpha-fetoprotein not only serves as a diagnostic marker for liver cancer, but also posses a variety of biological functions. However, the role of Alpha-fetoprotein on tumor angiogenesis and cell invasion remains incompletely understood. In this study, we aimed to evaluate if Alpha-fetoprotein can regulate the major angiogenic factors and matrix metalloproteinases in human liver cancer cells. Alpha-fetoprotein silencing was achieved by Stealth RNAi. Expression of Alpha-fetoprotein was examined by a full-automatic electrochemistry luminescence immunity analyzer. Expression of VEGF, VEGFR-2, MMP-9, and MMP-2 was examined by Western blot and immunocytochemistry. Apoptosis was detected by TUNEL assay. Angiogenesis was detected by in vitro angiogenesis assay kit. Silencing of Alpha-fetoprotein led to an increased apoptosis, which was associated with a decreased expression of vascular endothelial growth factor, vascular endothelial growth factor receptor 2, matrix metalloproteinases-2/9. These results suggest that Alpha-fetoprotein may play a regulatory role on angiogenesis and cell invasion during liver cancer development.
PMCID: PMC3938808  PMID: 24587407
13.  The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-Regulated by N-Myc 
PLoS ONE  2014;9(2):e88112.
The worst subtype of neuroblastoma is caused by MYCN oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed linc00467 gene expression through direct binding to the linc00467 gene promoter and reducing linc00467 promoter activity. While N-Myc suppressed the expression of RD3, the protein-coding gene immediately down-stream of linc00467 gene, through direct binding to the RD3 gene promoter and reducing RD3 promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.
PMCID: PMC3929584  PMID: 24586304
14.  Motor Cortex-Periaqueductal Gray-Spinal Cord Neuronal Circuitry May Involve in Modulation of Nociception: A Virally Mediated Transsynaptic Tracing Study in Spinally Transected Transgenic Mouse Model 
PLoS ONE  2014;9(2):e89486.
Several studies have shown that motor cortex stimulation provided pain relief by motor cortex plasticity and activating descending inhibitory pain control systems. Recent evidence indicated that the melanocortin-4 receptor (MC4R) in the periaqueductal gray played an important role in neuropathic pain. This study was designed to assess whether MC4R signaling existed in motor cortex- periaqueductal gray- spinal cord neuronal circuitry modulated the activity of sympathetic pathway by a virally mediated transsynaptic tracing study. Pseudorabies virus (PRV)-614 was injected into the left gastrocnemius muscle in adult male MC4R-green fluorescent protein (GFP) transgenic mice (n = 15). After a survival time of 4–6 days, the mice (n = 5) were randomly assigned to humanely sacrifice, and spinal cords and brains were removed and sectioned, and processed for PRV-614 visualization. Neurons involved in the efferent control of the left gastrocnemius muscle were identified following visualization of PRV-614 retrograde tracing. The neurochemical phenotype of MC4R-GFP-positive neurons was identified using fluorescence immunocytochemical labeling. PRV-614/MC4R-GFP dual labeled neurons were detected in spinal IML, periaqueductal gray and motor cortex. Our findings support the hypothesis that MC4R signaling in motor cortex-periaqueductal gray-spinal cord neural pathway may participate in the modulation of the melanocortin-sympathetic signaling and contribute to the descending modulation of nociceptive transmission, suggesting that MC4R signaling in motor cortex- periaqueductal gray-spinal cord neural pathway may modulate the activity of sympathetic outflow sensitive to nociceptive signals.
PMCID: PMC3929690  PMID: 24586817
15.  Efficient Delivery of Cyclic Peptides into Mammalian Cells with Short Sequence Motifs 
ACS chemical biology  2012;8(2):423-431.
Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is L-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7–13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2–5-fold higher than that of nonaarginine (R9). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities.
PMCID: PMC3574231  PMID: 23130658
cyclic peptide; cell-penetrating peptide; membrane permeability; membrane transporter; drug delivery
16.  Different types of atrial fibrillation, renal function, and mortality in elderly Chinese patients with coronary artery disease 
Atrial fibrillation (AF) is the most common arrhythmia in patients with chronic kidney disease (CKD), and the combined prevalence of these two disorders increases as the population ages. Both AF and CKD have risk factors for development of each other and eventual mortality. However, the relationship between different types of AF, CKD, and mortality remains unclear, especially in elderly Chinese patients with coronary artery disease.
This study comprised 1,050 patients of median age 86 (60–104) years with coronary artery disease. The end point was all-cause death during a mean follow-up of 417 days.
Of 219 patients identified to have AF, 128 had paroxysmal type, 44 had persistent type, and 47 had permanent type. After adjusting for confounders, the estimated glomerular filtration rate was lower and the prevalence of CKD was higher in patients with permanent AF but not in those with paroxysmal or persistent AF. During follow-up, 106 non-CKD patients and 112 CKD patients died; mortality was significantly higher in CKD patients with AF than in those without AF (36 [40.9%] versus 76 [26.8%]), but not in patients without CKD (17 [13.0%] versus 89 [16.3%]). In patients with CKD, paroxysmal AF was independently associated with higher mortality after adjustment but not persistent or permanent AF. No type of AF had an independent association with mortality in patients without CKD.
All types of AF had a high prevalence. Permanent AF was independently associated with an increased prevalence of CKD and a decreased estimated glomerular filtration rate. Paroxysmal AF was an independent risk factor for survival in patients with CKD but not in those without CKD.
PMCID: PMC3933252  PMID: 24600210
atrial fibrillation; chronic kidney disease; coronary artery disease; Chinese; elderly; mortality
17.  Triptolide inhibits MDM2 and induces apoptosis in acute lymphoblastic leukemia cells through a p53-independent pathway 
Molecular cancer therapeutics  2012;12(2):184-194.
Triptolide, a natural product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. The antitumor activity of triptolide is associated with its biological activities, as it inhibits various pro-proliferative or anti-apoptotic factors that are dominantly expressed in given types of cancer cells. Herein, we demonstrate that triptolide induced apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells overexpressing the MDM2 oncoprotein, by inhibiting MDM2 expression. More specifically, we found that triptolide inhibited MDM2 at the transcriptional level by suppressing its mRNA synthesis. This MDM2 inhibition led in turn to increased levels of p53 protein; however, p53 functionality was not activated, due to the fact that triptolide-treated cells lacked induction of p21 and PUMA as well as in G1 cell-cycle arrest. Triptolide-mediated downregulation of MDM2 increased inhibition of XIAP, its translational target, in a manner distinct from reactions to cellular stress and DNA-damaging agent ionizing radiation (IR) that induce XIAP due to p53-activated MDM2. These results suggest that increased inhibition of XIAP due to downregulation of MDM2 may play a critical role in triptolide-induced apoptosis in MDM2-overexpressing cancers.
PMCID: PMC3570632  PMID: 23243057
18.  Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach* 
Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.
PMCID: PMC3945918  PMID: 24443746
19.  Producing aglycons of ginsenosides in bakers' yeast 
Scientific Reports  2014;4:3698.
Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal plant that exhibits diverse pharmacological activities. Protopanaxadiol, protopanaxatriol and oleanolic acid are three basic aglycons of ginsenosides. Producing aglycons of ginsenosides in Saccharomyces cerevisiae was realized in this work and provides an alternative route compared to traditional extraction methods. Synthetic pathways of these three aglycons were constructed in S. cerevisiae by introducing β-amyrin synthase, oleanolic acid synthase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and NADPH-cytochrome P450 reductase from different plants. In addition, a truncated 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthase and 2,3-oxidosqualene synthase genes were overexpressed to increase the precursor supply for improving aglycon production. Strain GY-1 was obtained, which produced 17.2 mg/L protopanaxadiol, 15.9 mg/L protopanaxatriol and 21.4 mg/L oleanolic acid. The yeast strains engineered in this work can serve as the basis for creating an alternative way for producing ginsenosides in place of extractions from plant sources.
PMCID: PMC3892717  PMID: 24424342
20.  Chemical composition and in vitro evaluation of the cytotoxic and antioxidant activities of supercritical carbon dioxide extracts of pitaya (dragon fruit) peel 
Hylocereus polyrhizus and Hylocereus undatus are two varieties of the commonly called pitaya fruits, and pitaya fruits have gained popularity in many countries all over the world. However, studies on chemical composition and the nutritional quality of pitaya flesh peel are limited.
Extracts of pitaya (H. polyrhizus and H. undatus) peel were extracted by supercritical carbon dioxide extraction, and analyzed by gas chromatography–mass spectrometry analysis. Their cytotoxic and antioxidant activities were investigated. The main components of H. polyrhizus extract were β-amyrin (15.87%), α-amyrin (13.90%), octacosane (12.2%), γ-sitosterol (9.35%), octadecane (6.27%), 1-tetracosanol (5.19%), stigmast-4-en-3-one (4.65%), and campesterol (4.16%), whereas H. undatus were β-amyrin (23.39%), γ-sitosterol (19.32%), and octadecane (9.25%), heptacosane (5.52%), campesterol (5.27%), nonacosane (5.02%), and trichloroacetic acid, hexadecyl ester (5.21%). Both of the two extracts possessed good cytotoxic activities against PC3, Bcap-37, and MGC-803 cells (IC50 values ranging from 0.61 to 0.73 mg/mL), and the activities of their main components were also studied. Furthermore, these extracts also presented some radical scavenging activities, with IC50 values of 0.83 and 0.91 mg/mL, respectively.
This paper provides evidence for studying the chemical composition of supercritical carbon dioxide extracts of pitaya peel and their biological activity.
PMCID: PMC3880984  PMID: 24386928
21.  Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells 
As the function of transient receptor potential melastatin member 8 (TRPM8) in osteosarcoma is still unknown, we aim to investigate the possible effects and potential mechanisms of TRPM8 on cell proliferation, metastasis and chemosensitivity in osteosarcoma cells. We find that TRPM8 is aberrantly over-expressed in human osteosarcoma tissues and cell lines. Knockdown of TRPM8 by siRNA in osteosarcoma cells leads to the impaired regulation of intracellular Ca2+ concentration and then the Akt-GSK-3β pathway and the phosphorylation of p44/p42 and FAK are suppressed. Knockdown of TRPM8 not only negatively influences the cell proliferation and metastasis but also enhances epirubicin-induced cell apoptosis. Such results reveal that TRPM8 is worthy further investigation for its potential as a clinical biomarker and therapeutic target in osteosarcoma.
PMCID: PMC3879595  PMID: 24391455
TRPM8; osteosarcoma; epirubicin; apoptosis; MAPK
22.  Sensitivity of the discrete-time Kaplan-Meier estimate to nonignorable censoring: Application in a clinical trial 
Statistics in medicine  2012;31(25):10.1002/sim.5454.
Untestable assumptions about association between survival and censoring times can affect the validity of estimates of the survival distribution, including the Kaplan-Meier (KM) nonparametric MLE. This article explores the sensitivity of the KM curve to nonignorable censoring by extending the index of local sensitivity to nonignorability (ISNI) (Troxel et al. 2004, Zhang and Heitjan 2006) to the case of a nonparametric survival model. The method involves first specifying a coarse-data selection model to describe the association between the failure and censoring processes, then evaluating the slope of the nonparametric survival MLE ordinate with respect to a nonignorability parameter in the neighborhood of the ignorable model. We define the nonparametric MLE of the survival curve for a fixed value of the nonignorability parameter, and show in a simulation that ISNI analysis effectively captures local sensitivity to nonignorability. The method measures sensitivity in the sense of identifying functionals of the nonparametric MLE that nonignorability, if present, can affect substantially. We demonstrate the method with an application to a trial comparing mechanical assistance to optimal medical management in the treatment of end-stage heart failure.
PMCID: PMC3876928  PMID: 22806989
Coarse-data model; ignorability; informative censoring; ISNI; sensitivity analysis
23.  Variants of the PPARD Gene and Their Clinicopathological Significance in Colorectal Cancer 
PLoS ONE  2013;8(12):e83952.
Peroxisome proliferator-activated receptor delta (PPARD) is nuclear hormone receptor involved in colorectal cancer (CRC) differentiation and progression. The purpose of this study was to determine prevalence and spectrum of variants in the PPARD gene in CRC, and their contribution to clinicopathological endpoints.
Methods and Findings
Direct sequencing of the PPARD gene was performed in 303 primary tumors, in blood samples from 50 patients with ≥3 affected first-degree relatives, 50 patients with 2 affected first-degree relatives, 50 sporadic patients, 360 healthy controls, and in 6 colon cancer cell lines. Mutation analysis revealed 22 different transversions, 7 of them were novel. Three of all variants were somatic (c.548A>G, p.Y183C, c.425-9C>T, and c.628-16G>A). Two missense mutations (p.Y183C and p.R258Q) were pathogenic using in silico predictive program. Five recurrent variants were detected in/adjacent to the exons 4 (c.1-87T>C, c.1-67G>A, c.130+3G>A, and c.1-101-8C>T) and exon 7 (c.489T>C). Variant c.489C/C detected in tumors was correlated to worse differentiation (P = 0.0397).
We found 7 novel variants among 22 inherited or acquired PPARD variants. Somatic and/or missense variants detected in CRC patients are rare but indicate the clinical importance of the PPARD gene.
PMCID: PMC3877104  PMID: 24391853
24.  Identification and characterisation of non-coding small RNAs in the pathogenic filamentous fungus Trichophyton rubrum 
BMC Genomics  2013;14:931.
Accumulating evidence demonstrates that non-coding RNAs (ncRNAs) are indispensable components of many organisms and play important roles in cellular events, regulation, and development.
Here, we analysed the small non-coding RNA (ncRNA) transcriptome of Trichophyton rubrum by constructing and sequencing a cDNA library from conidia and mycelia. We identified 352 ncRNAs and their corresponding genomic loci. These ncRNA candidates included 198 entirely novel ncRNAs and 154 known ncRNAs classified as snRNAs, snoRNAs and other known ncRNAs. Further bioinformatic analysis detected 96 snoRNAs, including 56 snoRNAs that had been annotated in other organisms and 40 novel snoRNAs. All snoRNAs belonged to two major classes—C/D box snoRNAs and H/ACA snoRNAs—and their potential target sites in rRNAs and snRNAs were predicted. To analyse the evolutionary conservation of the ncRNAs in T. rubrum, we aligned all 352 ncRNAs to the genomes of six dermatophytes and to the NCBI non-redundant nucleotide database (NT). The results showed that most of the identified snRNAs were conserved in dermatophytes. Of the 352 ncRNAs, 102 also had genomic loci in other dermatophytes, and 27 were dermatophyte-specific.
Our systematic analysis may provide important clues to the function and evolution of ncRNAs in T. rubrum. These results also provide important information to complement the current annotation of the T. rubrum genome, which primarily comprises protein-coding genes.
PMCID: PMC3890542  PMID: 24377353
25.  Identifying ChIP-seq enrichment using MACS 
Nature protocols  2012;7(9):10.1038/nprot.2012.101.
Model-based Analysis of ChIP-seq (MACS) is a computational algorithm that identifies genome-wide locations of transcription/chromatin factor binding or histone modification from ChIP-seq data. MACS consists of four steps: removing redundant reads, adjusting read position, calculating peak enrichment, and estimating the empirical false discovery rate. In this protocol, we provide a detailed demonstration of how to install MACS and how to use it to analyze three common types of ChIP-seq datasets with different characteristics: the sequence-specific transcription factor FoxA1, the histone modification mark H3K4me3 with sharp enrichment, and the H3K36me3 mark with broad enrichment. We also explain how to interpret and visualize the results of MACS analyses. The algorithm requires approximately 3 GB of RAM and 1.5 hours of computing time to analyze a ChIP-seq dataset containing 30 million reads, an estimate that increases with sequence coverage. MACS is open-source and is available from
PMCID: PMC3868217  PMID: 22936215
MACS; ChIP-seq; peak calling; transcription factor; histone modification

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