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1.  A MicroRNA Signature Predicts Survival in Early Stage Small-Cell Lung Cancer Treated with Surgery and Adjuvant Chemotherapy 
PLoS ONE  2014;9(3):e91388.
Small-cell lung cancer (SCLC) is one of the most aggressive cancers, yet the molecular mechanisms underlying its devastating clinical outcome remain elusive. In this study, we investigated whether microRNA (miRNA) expression profiles can predict the clinical outcomes of SCLC patients. A total of 82 patients with limited SCLC, who were treated with surgical resection and adjuvant chemotherapy, were enrolled in this study. First, we surveyed the expression of 924 miRNAs from 42 SCLC patients to discover survival-relevant miRNAs and develop prognostic models, which were then validated in an independent cohort of 40 cases using quantitative real-time PCR. We found that the miR-150/miR-886-3p signature was significantly correlated with the overall survival (OS) of SCLC patients (p = 0.02) in the training set, and both miRNA expression levels were much lower in the SCLC samples than normal lung samples. The miRNA signature also proved to be a significant predictor of survival in the validation set. Patients with high-risk miRNA signatures had poor overall survival (p = 0.005) and progression-free survival (p = 0.017) compared with those with low-risk scores. These findings retained statistical significance after adjusting for age, gender and smoking status (HR: 0.26, 95%: CI 0.10–0.69, p = 0.007), which suggested it may be an independent predictor of survival. In summary, we developed a prognostic miR-150/miR-886-3p signature and validated expression in an independent dataset of resectable SCLC. These preliminary results indicated that miRNAs may serve as promising molecular prognostic markers and new therapeutic targets for SCLC.
PMCID: PMC3956664  PMID: 24637927
2.  Detection and Analysis of Human Papillomavirus (HPV) DNA in Breast Cancer Patients by an Effective Method of HPV Capture 
PLoS ONE  2014;9(3):e90343.
Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16(HPV16) was detected in 1/7, 1/7, 1/7and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer.
PMCID: PMC3948675  PMID: 24614680
3.  βig-h3 Promotes Human Osteosarcoma Cells Metastasis by Interacting with Integrin α2β1 and Activating PI3K Signaling Pathway 
PLoS ONE  2014;9(3):e90220.
Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment.
PMCID: PMC3942417  PMID: 24595049
Cancer is the second leading cause of death in the US. Dietary factors account for at least 30% of all cancers in Western countries. Since people do not consume individual foods but rather combinations of them, the assessment of dietary patterns may offer valuable information when determining associations between diet and cancer risk.
We examined the association between dietary patterns (non-vegetarians, lacto, pesco, vegan, and semi-vegetarian) and the overall cancer incidence among 69,120 participants of the Adventist Health Study-2. Cancer cases were identified by matching to cancer registries. Cox-proportional hazard regression analysis was performed to estimate hazard ratios, with “attained age” as the time variable.
2,939 incident cancer cases were identified. The multivariate HR of overall cancer risk among vegetarians compared to non-vegetarians was statistically significant (HR=0.92; 95%CI: 0.85, 0.99) for both genders combined. Also, a statistically significant association was found between vegetarian diet and cancers of the gastrointestinal tract (HR=0.76; 95%CI: 0.63, 0.90). When analyzing the association of specific vegetarian dietary patterns, vegan diets showed statistically significant protection for overall cancer incidence (HR=0.84; 95%CI: 0.72, 0.99) in both genders combined and for female-specific cancers (HR=0.66; 95%CI: 0.47, 0.92). Lacto-ovo-vegetarians appeared to be associated with decreased risk of cancers of the gastrointestinal system (HR=0.75; 95%CI: 0.60, 0.92).
Vegetarian diets seem to confer protection against cancer.
Vegan diet seems to confer lower risk for overall and female-specific cancer compared to other dietary patterns. The lacto-ovo-vegetarian diets seem to confer protection from cancers of the gastrointestinal tract.
PMCID: PMC3565018  PMID: 23169929
cancer; vegetarian dietary patterns; diet; vegan; lacto-ovo-vegetarians
5.  Overexpression of 14-3-3θ promotes tumor metastasis and indicates poor prognosis in breast carcinoma 
Oncotarget  2013;5(1):249-257.
An isoform of the 14-3-3 protein family, 14-3-3θ has been linked with tumor cell proliferation and apoptosis. However, the role of 14-3-3θ in the progression of breast cancer remains unknown. Here, we report that 14-3-3θ plays a critical role in breast cancer metastasis and prognosis. The expression of 14-3-3θ was markedly higher in breast cancer tissues compared to adjacent normal tissues. A hospital-based study cohort of 216 breast cancer patients was evaluated in this study. The level of 14-3-3θ expression was determined and correlated based upon tumor clinicopathological features, disease-free survival, and overall survival. We found that overexpression of 14-3-3θ was correlated with advanced TNM stage (P < 0.05), lymph node metastasis (P < 0.05), and ER negative status (P < 0.05). Breast cancer patients with high 14-3-3θ expression had a shorter overall survival and a higher rate of recurrence than those with low 14-3-3θ expression. Additionally, knockdown of 14-3-3θ expression in breast cancer cells inhibited metastasis in vitro. Similarly, an in vivo assay showed that 14-3-3θ knockdown dramatically suppressed the growth of breast cancer xenografts and inhibited tumor cell metastasis in a lung metastasis model. Thus, this study provided the first evidence that 14-3-3θ is a novel tumor suppressor and may serve as a candidate prognostic biomarker and target for new therapies in metastatic breast cancer.
PMCID: PMC3960205  PMID: 24371149
14-3-3θ; Breast Cancer; Metastasis; Prognosis
6.  Decreasing Levels of the cdk5 Activators, p25 and p35, Reduces Excitotoxicity in Striatal Neurons 
Journal of Huntington's disease  2012;1(1):10.3233/JHD-2012-129000.
Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG trinucleotide repeat sequence in the huntingtin gene. The resulting poly-glutamine expansion in the huntingtin protein imparts a novel toxic gain of function causing selective loss of medium spiny neurons (MSNs) in the striatum. Although the exact mechanism of cell death is unclear, recent evidence suggests involvement of NMDA-receptor mediated excitotoxicity and aberrant cyclin dependent kinase 5 (cdk5) activity in striatal cells undergoing neurodegeneration. In this study we directly tested the effect of reduced levels of p25 and p35, two proteins required for cdk5 activation, on striatal neurodegeneration using mice with targeted deletion of p35. Quinolinic acid (QA) injected into the striatum of mice causes NMDA-receptor mediated cell death, and these QA-induced striatal lesions were examined in p35 hemizygous null (p35+/−) and wildtype (WT) mice. Striatal QA lesion volumes were 30% smaller in p35+/− mice than in WT mice. Furthermore, primary neuronal cultures of MSNs from P0 p35+/− pups displayed 33% less apoptotic neurons following NMDA treatment than those from WT pups. Examination of YAC128 mouse model of HD showed elevated p25 levels in striatum following intrastriatal QA injection. Our findings provide direct evidence for p25 and p35 involvement in excitotoxic neurodegeneration of MSNs and suggest a role for the cdk5 pathway in HD striatal neurodegeneration.
PMCID: PMC3864911  PMID: 24353748
Huntington’s disease; medium spiny neuron; striatum; YAC128; cdk5; p25; quinolinic acid; NMDA; excitotoxic; neurodegeneration
7.  Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia 
The impact of oncogene activation and hypoxia on energy metabolism is analyzed by integrating quantitative measurements into a redox-balanced metabolic flux model. Glutamine-driven oxidative phosphorylation is found to be a major ATP source even in oncogene-expressing or hypoxic cells.
The integration of oxygen uptake measurements and LC-MS-based isotope tracer analyses in a redox-balanced metabolic flux model enabled quantitative determination of energy generation pathways in cultured cells.In transformed mammalian cells, even in hypoxia (1% oxygen), oxidative phosphorylation produces the majority of ATP.The oncogene Ras simultaneously increases glycolysis and decreases oxidative phosphorylation, thus resulting in no net increase in ATP production.Glutamine is the major source of high-energy electrons for oxidative phosphorylation, especially upon Ras activation.
Mammalian cells can generate ATP via glycolysis or mitochondrial respiration. Oncogene activation and hypoxia promote glycolysis and lactate secretion. The significance of these metabolic changes to ATP production remains however ill defined. Here, we integrate LC-MS-based isotope tracer studies with oxygen uptake measurements in a quantitative redox-balanced metabolic flux model of mammalian cellular metabolism. We then apply this approach to assess the impact of Ras and Akt activation and hypoxia on energy metabolism. Both oncogene activation and hypoxia induce roughly a twofold increase in glycolytic flux. Ras activation and hypoxia also strongly decrease glucose oxidation. Oxidative phosphorylation, powered substantially by glutamine-driven TCA turning, however, persists and accounts for the majority of ATP production. Consistent with this, in all cases, pharmacological inhibition of oxidative phosphorylation markedly reduces energy charge, and glutamine but not glucose removal markedly lowers oxygen uptake. Thus, glutamine-driven oxidative phosphorylation is a major means of ATP production even in hypoxic cancer cells.
PMCID: PMC3882799  PMID: 24301801
cancer bioenergetics; isotope tracing; metabolic flux analysis
8.  A Subanesthetic Dose of Isoflurane during Postconditioning Ameliorates Zymosan-Induced Neutrophil Inflammation Lung Injury and Mortality in Mice 
Mediators of Inflammation  2013;2013:479628.
Anesthetic isoflurane (ISO) has immunomodulatory effects. In the present study, we investigated whether a subanesthetic dose of ISO (0.7%) protected against zymosan (ZY) induced inflammatory responses in the murine lung and isolated neutrophils. At 1 and 6 hrs after ZY administration intraperitoneally, ISO was inhaled for 1 hr, and 24 hrs later, lung inflammation and injury were assessed. We found that ISO improved the survival rate of mice and mitigated lung injury as characterized by the histopathology, wet-to-dry weight ratio, protein leakage, and lung function index. ISO significantly attenuated ZY-induced lung neutrophil recruitment and inflammation. This was suggested by the downregulation of (a) endothelial adhesion molecule expression and myeloperoxidase (MPO) activity in lung tissue and polymorphonuclear neutrophils (b) chemokines, and (c) proinflammatory cytokines in BALF. Furthermore, ZY-induced nuclear translocation and DNA-binding activity of NF-κB p65 were also reduced by ISO. ISO treatment inhibited iNOS expression and activity, as well as subsequent nitric oxide generation. Consistent with these in vivo observations, in vitro studies confirmed that ISO blocked NF-κB and iNOS activation in primary mouse neutrophils challenged by ZY. These results provide evidence that 0.7% ISO ameliorates inflammatory responses in ZY-treated mouse lung and primary neutrophils.
PMCID: PMC3863458  PMID: 24369446
9.  The Puzzle of Italian Rice Origin and Evolution: Determining Genetic Divergence and Affinity of Rice Germplasm from Italy and Asia 
PLoS ONE  2013;8(11):e80351.
The characterization of genetic divergence and relationships of a set of germplasm is essential for its efficient applications in crop breeding and understanding of the origin/evolution of crop varieties from a given geographical region. As the largest rice producing country in Europe, Italy holds rice germplasm with abundant genetic diversity. Although Italian rice varieties and the traditional ones in particular have played important roles in rice production and breeding, knowledge concerning the origin and evolution of Italian traditional varieties is still limited. To solve the puzzle of Italian rice origin, we characterized genetic divergence and relationships of 348 rice varieties from Italy and Asia based on the polymorphisms of microsatellite fingerprints. We also included common wild rice O. rufipogon as a reference in the characterization. Results indicated relatively rich genetic diversity (He = 0.63-0.65) in Italian rice varieties. Further analyses revealed a close genetic relationship of the Italian traditional varieties with those from northern China, which provides strong genetic evidence for tracing the possible origin of early established rice varieties in Italy. These findings have significant implications for the rice breeding programs, in which appropriate germplasm can be selected from a given region and utilized for transferring unique genetic traits based on its genetic diversity and evolutionary relationships.
PMCID: PMC3827184  PMID: 24265814
10.  Validation of the European Organization for Research and Treatment of Cancer cervical cancer module for Chinese patients with cervical cancer 
The aim of our study was to assess, for the first time, the validity, reliability, and acceptability of the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life questionnaire (QLQ) cervical cancer module (CX24) in Chinese cervical cancer patients.
Patients and methods
One hundred fifteen outpatients with cervical cancer in the First Affiliated Hospital of Xinxiang Medical University from May 2013 to July 2013 were included in this study. All participants self-administered the EORTC QLQ-CX24 and the core questionnaire (EORTC QLQ-C30), and the Karnofsky Performance Scale was performed to evaluate scores. Data were analyzed with Cronbach’s α coefficient, Pearson correlation test, multitrait scaling analysis, and Mann–Whitney U test.
Scale reliability was confirmed by Cronbach’s α coefficients for internal consistency, which ranged from 0.71 to 0.82. Convergent and discriminant validity were confirmed by multitrait scaling analysis, which revealed three (3.4%) scaling errors for symptom experience scales and zero (0%) for body image as well as sexual/vaginal functioning scales. Higher missing value rate occurred in sexuality-related items. The clinical validity of the Chinese version of the EORTC QLQ-CX24 was demonstrated by the ability to discriminate among patients in different International Federation of Gynecology and Obstetrics stages.
The EORTC QLQ-CX24 was proved to be a reliable and valid instrument with which to measure the quality of life in cervical cancer patients in the People’s Republic of China.
PMCID: PMC3804534  PMID: 24204125
cervical cancer; quality of life; EORTC QLQ-CX24; People’s Republic of China
11.  Palliative surgery for primary sarcoma in the abdominal aorta: A case report and review of the literature 
Oncology Letters  2013;6(6):1738-1740.
Primary sarcoma of the aorta is extremely rare and accounts for <1% of all sarcomas. The present study describes the case of a 45-year-old male who presented with lower limb and abdominal pain. Abdominal computed tomography (CT) and magnetic resonance (MR) arteriography revealed a tumor that extended from the infrarenal aorta to the aortic bifurcation. The external and internal iliac arteries were occluded by the tumor incursion. Palliative surgery was performed for the sarcoma since the patient refused a radical resection. To improve the blood supply to the lower limbs, an axillary bifemoral bypass was established. Following the surgery, the pain was significantly reduced. However, the patient succumbed due to extensive metastasis 6 months after this surgery. Aortic sarcoma is an extremely rare disease with a poor prognosis. A diagnosis at a relatively early stage is necessary for a longer survival time. Radical surgery is the most significant treatment. Patients at advanced stages should consider palliative surgery in order to improve their quality of life.
PMCID: PMC3834868  PMID: 24273607
abdominal aortic aneurysm; palliative surgery
12.  A quantitative analytic pipeline for evaluating neuronal activities by high throughput synaptic vesicle imaging 
NeuroImage  2012;62(3):2040-2054.
Synaptic vesicle dynamics play an important role in the study of neuronal and synaptic activities of neurodegradation diseases ranging from the epidemic Alzheimer’s disease to the rare Rett syndrome. A high-throughput assay with a large population of neurons would be useful and efficient to characterize neuronal activity based on the dynamics of synaptic vesicles for the study of mechanisms or to discover drug candidates for neurodegenerative and neurodevelopmental disorders. However, the massive amounts of image data generated via high throughput screening require enormous manual processing time and effort, restricting the practical use of such an assay. This paper presents an automated analytic system to process and interpret the huge data set generated by such assays. Our system enables the automated detection, segmentation, quantification, and measurement of neuron activities based on the synaptic vesicle assay. To overcome challenges such as noisy background, inhomogeneity, and tiny object size, we first employ MSVST (Multi-Scale Variance Stabilizing Transform) to obtain a denoised and enhanced map of the original image data. Then, we propose an adaptive thresholding strategy to solve the inhomogeneity issue, based on the local information, and to accurately segment synaptic vesicles. We design algorithms to address the issue of tiny objects-of-interest overlapping. Several post-processing criteria are defined to filter false positives. A total of 152 features are extracted for each detected vesicle. A score is defined for each synaptic vesicle image to quantify the neuron activity. We also compare the unsupervised strategy with the supervised method. Our experiments on hippocampal neuron assays showed that the proposed system can automatically detect vesicles and quantify their dynamics for evaluating neuron activities. The availability of such an automated system will open opportunities for investigation of synaptic neuropathology and identification of candidate therapeutics for neurodegeneration.
PMCID: PMC3437259  PMID: 22732566
synaptic vesicle; neuron activity; detection and quantification; neurodegenerative disease; high throughput image screening
13.  Biases Introduced by Choosing Controls to Match Risk Factors of Cases in Biomarker Research 
Clinical chemistry  2012;58(8):1242-1251.
Selecting controls that match cases on risk factors for the outcome is a pervasive practice in biomarker research studies. Yet, such matching biases estimates of biomarker prediction performance. The magnitudes of bias are unknown.
We examined the prediction performance of biomarkers and improvements in prediction gained by adding biomarkers to risk factor information. Data simulated from bivariate normal statistical models and data from a study to identify critically ill patients were used. We compared true performance with that estimated from case-control studies that do or do not use matching. Receiver operating characteristic curves quantified performance. We propose a new statistical method to estimate prediction performance from matched studies when data on the matching factors are available for subjects in the population.
Performance estimated with standard analyses can be grossly biased by matching especially when biomarkers are highly correlated with matching risk factors. In our studies, the performance of the biomarker alone was underestimated while the improvement in performance gained by adding the marker to risk factors was overestimated by 2 to 10 fold. We found examples where the relative ranking of two biomarkers for prediction was inappropriately reversed by use of a matched design. The new approach to estimation corrected for bias in matched studies.
To properly gauge prediction performance in the population or the improvement gained by adding a biomarker to known risk factors, matched case-control studies must be supplemented with risk factor information from the population and must be analyzed with nonstandard statistical methods.
PMCID: PMC3464972  PMID: 22730452
design; diagnosis; prediction; prognosis; receiver operating characteristic curve
14.  The Efficacy and Safety of Nucleos(t)ide Analogues in Patients with Spontaneous Acute Exacerbation of Chronic Hepatitis B: A Systematic Review and Meta-Analysis 
PLoS ONE  2013;8(6):e65952.
Spontaneous acute exacerbation (AE) of chronic hepatitis B (CHB) is often detrimental but sometimes leads to sustained immune control and disease remission. The efficacy and safety of nucleos(t)ide analogues (NAs) in patients with spontaneous AE of CHB remains unclear.
We performed a systematic review and meta-analysis of NAs in patients with spontaneous AE of CHB. We calculated pooled effects of NAs in these patients of each study and conducted quantitative meta-analysis, displaying results using Forest plots.
15 studies were included and substantial heterogeneity was noted in the inclusion/exclusion criteria and controls. Pooled data showed no benefit of lamivudine (LAM) vs. untreated controls for transplant-free survival in patients with spontaneous AE of CHB (OR = 0.98 (95% CI, 0.50–1.92; P = 0.956)), hepatic decompensation (OR = 0.94 (95% CI, 0.47–1.88; P = 0.862)) and liver failure owing to AE (OR = 2.30 (95% CI, 0.35–15.37; P = 0.387)) at 3 months. Entecavir achieved even higher short-term mortality than LAM. NAs led to rates of ALT normalization, undetectable HBV DNA, HBeAg loss, HBeAg seroconversion and drug resistance at 1 year in 88%, 61%, 46%, 35% and 5%. Pooled data also showed benefit favoring LAM vs. untreated controls for ALT normalization (OR = 1.98 (95% CI, 1.03–3.80; P = 0.039)) and undetectable HBV DNA (OR = 38.50 (95% CI, 7.68–192.99; P<0.001)) at 3 months. All NAs were relatively safe and well tolerated.
NAs had no obvious impact on short-term survival in patients with AE of CHB, despite of possible better antiviral responses. We suggest additional studies to evaluate the efficacy of other NAs and early introduction of immunosuppressant in combination with NAs. We highlight developing prognostic models to identify predictors of mortality and disease progression for AE of CHB.
PMCID: PMC3679018  PMID: 23776577
15.  Inhibition of NADPH Oxidase Mediates Protective Effect of Cardiotonic Pills against Rat Heart Ischemia/Reperfusion Injury 
Cardiotonic pill (CP) is a compound Chinese medicine currently used in China for treatment of ischemic angina pectoris. Our previous results indicated that a single dosing of CP pretreatment at 0.8 g/kg attenuates ischemia/reperfusion- (I/R-) induced myocardial injury and cardiac microcirculatory disturbance. The present study aimed to investigate the effect of CP at low dosage in a multiple dosing manner and to uncover the mechanism of antioxidative activity of CP. Male Sprague-Dawley rats were subjected to left anterior descending artery occlusion for 30 min followed by 60 min reperfusion. CP was administrated daily by gavage for six days at 0.1, 0.4, and 0.8 g/kg/day before I/R. Results showed that multiple dosing of CP at three doses significantly reduced I/R-induced myocardial injury, microcirculatory disturbance, and oxidative stress. CP dramatically inhibited I/R-induced nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase subunit gp91phox expression and p67phox and p47phox translocation from cytosol to cell membrane. Translocation of cytosolic subunits to membrane is required for the activation of NADPH oxidase. These data suggested that multiple dosing of CP at doses ranging from 0.1 to 0.8 g/kg/day reduced I/R-induced rat myocardial injury and microcirculatory disturbance, which was mediated by inhibition of NADPH oxidase activation.
PMCID: PMC3690747  PMID: 23840265
16.  Huang Qi Jian Zhong Pellet Attenuates TNBS-Induced Colitis in Rats via Mechanisms Involving Improvement of Energy Metabolism 
Huang Qi Jian Zhong Pellet (HQJZ) is a famous Chinese medicine formula for treatment of various gastrointestinal tract diseases. This study investigated the role of HQJZ in 2,4,6-trinitrobenzene sulfonic acid- (TNBS-) induced colitis and its underlying mechanism. Colonic mucosal injury was induced by TNBS in the Sprague-Dawley rats. In the HQJZ treatment group, HQJZ was administered (2 g/kg) for 14 days starting from day 1 after TNBS infusion. Colonic mucosal injury occurred obviously 1 day after TNBS challenge and did not recover distinctively until day 15, including an increase in macro- and microscopic scores, a colonic weight index, a decrease in colonic length, a number of functional capillaries, and blood flow. Inverted intravital microscopy and ELISA showed colonic microcirculatory disturbances and inflammatory responses after TNBS stimulation, respectively. TNBS decreased occludin, RhoA, and ROCK-I, while increasing Rac-1, PAK-1, and phosphorylated myosin light chain. In addition, ATP content and ATP5D expression in colonic mucosa decreased after TNBS challenge. Impressively, treatment with HQJZ significantly attenuated all of the alterations evoked by TNBS, promoting the recovery of colonic injury. The present study demonstrated HQJZ as a multitargeting management for colonic mucosal injury, which set in motion mechanisms involving improvement of energy metabolism.
PMCID: PMC3690262  PMID: 23840258
17.  Genome-Wide RNAi Screening Identifies Genes Inhibiting the Migration of Glioblastoma Cells 
PLoS ONE  2013;8(4):e61915.
Glioblastoma Multiforme (GBM) cells are highly invasive, infiltrating into the surrounding normal brain tissue, making it impossible to completely eradicate GBM tumors by surgery or radiation. Increasing evidence also shows that these migratory cells are highly resistant to cytotoxic reagents, but decreasing their migratory capability can re-sensitize them to chemotherapy. These evidences suggest that the migratory cell population may serve as a better therapeutic target for more effective treatment of GBM. In order to understand the regulatory mechanism underlying the motile phenotype, we carried out a genome-wide RNAi screen for genes inhibiting the migration of GBM cells. The screening identified a total of twenty-five primary hits; seven of them were confirmed by secondary screening. Further study showed that three of the genes, FLNA, KHSRP and HCFC1, also functioned in vivo, and knocking them down caused multifocal tumor in a mouse model. Interestingly, two genes, KHSRP and HCFC1, were also found to be correlated with the clinical outcome of GBM patients. These two genes have not been previously associated with cell migration.
PMCID: PMC3625150  PMID: 23593504
18.  Identification of SNP-containing regulatory motifs in the myelodysplastic syndromes model using SNP arrays and gene expression arrays 
Chinese Journal of Cancer  2013;32(4):170-185.
Myelodysplastic syndromes have increased in frequency and incidence in the American population, but patient prognosis has not significantly improved over the last decade. Such improvements could be realized if biomarkers for accurate diagnosis and prognostic stratification were successfully identified. In this study, we propose a method that associates two state-of-the-art array technologies—single nucleotide polymorphism (SNP) array and gene expression array—with gene motifs considered transcription factor-binding sites (TFBS). We are particularly interested in SNP-containing motifs introduced by genetic variation and mutation as TFBS. The potential regulation of SNP-containing motifs affects only when certain mutations occur. These motifs can be identified from a group of co-expressed genes with copy number variation. Then, we used a sliding window to identify motif candidates near SNPs on gene sequences. The candidates were filtered by coarse thresholding and fine statistical testing. Using the regression-based LARS-EN algorithm and a level-wise sequence combination procedure, we identified 28 SNP-containing motifs as candidate TFBS. We confirmed 21 of the 28 motifs with ChIP-chip fragments in the TRANSFAC database. Another six motifs were validated by TRANSFAC via searching binding fragments on co-regulated genes. The identified motifs and their location genes can be considered potential biomarkers for myelodysplastic syndromes. Thus, our proposed method, a novel strategy for associating two data categories, is capable of integrating information from different sources to identify reliable candidate regulatory SNP-containing motifs introduced by genetic variation and mutation.
PMCID: PMC3845573  PMID: 23327800
Association study; genetic variation and mutation; transcription factor-binding sites; myelodysplastic syndromes
19.  Bis{1-[(1H-benzimidazol-1-yl)meth­yl]-1H-imidazole-κN 3}bis­(3,5-dicarb­oxy­benzoato-κ2 O 1,O 1′)nickel(II) octa­hydrate 
In the title complex, [Ni(C9H5O6)2(C11H10N4)2]·8H2O, the NiII ion exhibits site symmetry 2. It has a distorted octa­hedral coordination defined by two N atoms from two symmetry-related 1-[(1H-benzimidazol-1-yl)meth­yl]-1H-imidazole ligands and four O atoms from two symmetry-related 3,5-dicarb­oxy­benzoate anions. In the crystal, the complex mol­ecules and solvent water mol­ecules are linked via O—H⋯O, O—H⋯N and N—H⋯O hydrogen bonds, forming a three-dimensional structure. There are also a number of C—H⋯O inter­actions present.
PMCID: PMC3629470  PMID: 23633988
20.  QiShenYiQi Pills, a Compound Chinese Medicine, Ameliorates Doxorubicin-Induced Myocardial Structure Damage and Cardiac Dysfunction in Rats 
QiShenYiQi Pills (QSYQ) is a compound Chinese medicine used for treatment of cardiovascular diseases. The present study investigated the effects of QSYQ on the Doxorubicin- (DOX-) induced disorders in rat cardiac structure and function and the possible mechanism underlying. A total of 24 male Sprague-Dawley rats were administrated by intraperitoneal injections with DOX at a dose of 2.5 mg/kg, once every day for a total of 6 times. After the 6th injection, the rats were evaluated by echocardiographic analysis, and the animals with injured heart (n = 14) were divided into 2 groups and further treated with (n = 7) or without (n = 7) QSYQ by gavage at a dose of 0.2 g/day, once a day, over the next 2 weeks. Two weeks after QSYQ treatment, the following variables were assessed: myocardial blood flow (MBF) by Laser-Doppler Perfusion Imager, the ratio of heart weight to body weight (HW/BW), myocardial histology, myocardial content of ATP, AMP, free fatty acids (FFAs) and AMP/ATP by ELISA, and expression of PPARα, PGC-1α, and ATP 5D by Western blot. Statistical analysis was performed using one-way ANOVA followed by Turkey test for multiple comparisons. DOX challenge significantly increased left ventricular internal diameter and HW/BW and decreased the thickness of the left ventricular posterior wall, the left ventricle ejection fraction, and the left ventricle fractional shortening. DOX also increased AMP, FFA, and AMP/ATP, decreased ATP, and downregulated the protein content of ATP 5D, PPAR α, and PGC-1 α. All these DOX-induced cardiac insults were attenuated significantly by QSYQ treatment. These results show the potential of QSYQ to ameliorate DOX-induced disorders in cardiac structure and function; this effect may be related to the increase in myocardial ATP content via the upregulation of ATP 5D, PPAR α, and PGC-1 α and the oxidation of FFA.
PMCID: PMC3600323  PMID: 23533487
21.  Computational Approach to Seasonal Changes of Living Leaves 
This paper proposes a computational approach to seasonal changes of living leaves by combining the geometric deformations and textural color changes. The geometric model of a leaf is generated by triangulating the scanned image of a leaf using an optimized mesh. The triangular mesh of the leaf is deformed by the improved mass-spring model, while the deformation is controlled by setting different mass values for the vertices on the leaf model. In order to adaptively control the deformation of different regions in the leaf, the mass values of vertices are set to be in proportion to the pixels' intensities of the corresponding user-specified grayscale mask map. The geometric deformations as well as the textural color changes of a leaf are used to simulate the seasonal changing process of leaves based on Markov chain model with different environmental parameters including temperature, humidness, and time. Experimental results show that the method successfully simulates the seasonal changes of leaves.
PMCID: PMC3596921  PMID: 23533545
22.  Protection by Salidroside against Bone Loss via Inhibition of Oxidative Stress and Bone-Resorbing Mediators 
PLoS ONE  2013;8(2):e57251.
Oxidative stress is a pivotal pathogenic factor for bone loss in mouse model. Salidroside, a phenylpropanoid glycoside extracted from Rhodiola rosea L, exhibits potent antioxidative effects. In the present study, we used an in vitro oxidative stress model induced by hydrogen peroxide (H2O2) in MC3T3-E1 cells and a murine ovariectomized (OVX) osteoporosis model to investigate the protective effects of salidroside on bone loss and the related mechanisms. We demonstrated that salidroside caused a significant (P<0.05) elevation of cell survival, alkaline phosphatase (ALP) staining and activity, calcium deposition, and the transcriptional expression of Alp, Col1a1 and Osteocalcin (Ocn) in the presence of H2O2. Moreover, salidroside decreased the production of intracellular reactive oxygen species (ROS), and osteoclast differentiation inducing factors such as receptor activator of nuclear factor-kB ligand (RANKL) and IL-6 induced by H2O2. In vivo studies further demonstrated that salidroside supplementation for 3 months caused a decrease in malondialdehyde (MDA) and an increase in reduced glutathione (GSH) concentration in blood of ovariectomized mouse (P<0.05), it also improved trabecular bone microarchitecture and bone mineral density in the fourth lumbar vertebra and distal femur. Our study indicated that the protection provided by salidroside in alleviating bone loss was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage to bone-forming cells, suggesting that salidroside can be used as an effective remedy in the treatment or prevention of osteoporosis.
PMCID: PMC3577746  PMID: 23437352
23.  Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation 
PLoS ONE  2013;8(2):e56142.
Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTDMi, the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTDMi is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTDMi could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.
PMCID: PMC3571961  PMID: 23418525
24.  Efficient selection and evaluation of transgenic lines of Crambe abyssinica 
Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L−1 resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L−1. Based on this result, the hygromycin concentration up to 10 mg L−1 was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 μg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 μl enzyme was recomended for a complete digestion of up to 80 μg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.
PMCID: PMC3664313  PMID: 23750164
Crambe abyssinica; genetic transformation; hygromycin selection; qRT-PCR; Southern blotting
25.  A Computational model for compressed sensing RNAi cellular screening 
BMC Bioinformatics  2012;13:337.
RNA interference (RNAi) becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive.
In this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi), which employs a unique combination of group of small interfering RNAs (siRNAs) to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs) and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear), which is ill-posed in general. However, the recently developed compressed sensing (CS) theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially.
This csRNAi system is very promising in saving both time and cost for large-scale RNAi screening experiments which may benefit the biological research with respect to cellular processes and pathways.
PMCID: PMC3544734  PMID: 23270311

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