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1.  Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation 
Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure.
We found that piperazine-N,N’-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging.
We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0056-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4320506  PMID: 25645690
PIPES; HEPES; Artifact; Inter-rhabdomeral space; Rhabdomere; Eyes shut; Drosophila; Photoreceptor; Biological tube
2.  Association of prediagnostic physical activity with survival following breast cancer diagnosis: influence of TP53 mutation status 
Cancer causes & control : CCC  2013;24(12):10.1007/s10552-013-0294-x.
Physical activity both before and after breast cancer diagnosis has been associated with improved survival. However, it is not clear whether this association differs by molecular features of the tumor or by recency of the physical activity to the time of diagnosis.
We examined the association of prediagnostic physical activity with survival in a cohort of 1,170 women with primary, incident, and histologically confirmed breast cancer, examining tumor molecular subtypes. Cox regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI).
Mean follow-up time was 87.4 months after breast cancer diagnosis; there were 170 deaths identified. Compared with inactive patients (<3 hours/ week), women with higher average lifetime physical activity (>6 hours/ week) had reduced risk of all-cause mortality (adjusted HR = 0.61, 95% CI, 0.40-0.95; p trend = 0.04). There were no clear differences in the associations for lifetime and more recent physical activity. Lifetime physical activity was also weakly associated with decreased risk of breast cancer-specific mortality. Higher lifetime physical activity was associated with reduced risk of all-cause mortality among women with ER-positive tumors (HR = 0.52, 95% CI, 0.29-0.93), and mutant TP53 tumors (HR = 0.22, 95% CI, 0.06-0.72); however, no statistically significant interactions were observed for ER or TP53 status.
Our study further supports that prediagnostic physical activity improves overall survival following breast cancer, and suggests that the associations of prediagnostic physical activity with survival following breast cancer may vary by molecular features of the tumor, particularly ER and TP53 status.
PMCID: PMC3860883  PMID: 24068557
physical activity; breast cancer survival; TP53 mutation; epidemiology
3.  Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein-Protein Interactions 
Analytical chemistry  2013;85(20):10.1021/ac4023082.
Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods.
PMCID: PMC3845662  PMID: 24060167
4.  The Actomyosin Machinery Is Required for Drosophila Retinal Lumen Formation 
PLoS Genetics  2014;10(9):e1004608.
Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.
Author Summary
Biological tubes are integral units of tissues and organs such as lung, kidney, and the cardiovascular system. The fundamental design of tubes involves a central lumen wrapped by a sheet of cells. To function properly, the tubes require a precise genetic control over their creation, the diametric growth and maintenance of the lumen during development. In the fruit fly, Drosophila melanogaster, the photoreceptor cells of the eye form a tubular structure. The formation of the retinal lumen is critical for separating and positioning the light sensing organelles of each photoreceptor cell to achieve visual sensitivity. In an effort to investigate the mechanisms of Drosophila retinal lumen formation, we identified a contractile machinery that was present at the apical portion of photoreceptor cells. Our data is consistent with the idea that a contractile force contributes to the initial separation of the juxtaposed apical membranes and subsequent enlargement of the luminal space. Our work suggests that building a biological tube requires not only an extrinsic pushing force provided by the growing central lumen, but also a cell intrinsic pulling force powered by contraction of cells lining the lumen. Our findings expand and demonstrate the coordination of several molecular mechanisms to generate a tube.
PMCID: PMC4168998  PMID: 25233220
5.  Pregnancy-related Characteristics and Breast Cancer Risk 
Cancer causes & control : CCC  2013;24(9):1675-1685.
Breast tissues undergo extensive physiologic changes during pregnancy, which may affect breast carcinogenesis. Gestational hypertension, pre-eclampsia/eclampsia, gestational diabetes, pregnancy weight gain, and nausea and vomiting (N&V) during pregnancy may be indicative of altered hormonal and metabolic profiles and could impact breast cancer risk. Here, we examined associations between these characteristics of a woman’s pregnancy and her subsequent breast cancer risk. Participants were parous women that were recruited to a population-based case-control study (Western New York Exposures and Breast Cancer Study). Cases (n=960), aged 35-79 years, had incident, primary, histologically-confirmed breast cancer. Controls (n=1,852) were randomly selected from Motor Vehicle records (<65 years) or Medicare rolls (≥65 years). Women were queried on their lifetime pregnancy experiences. Multivariable-adjusted logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). N&V during pregnancy was inversely associated with breast cancer risk. Relative to those who never experienced N&V, ever experiencing N&V was associated with decreased risk (OR 0.69, 95% CI: 0.56-0.84) as were increased N&V severity (P-trend<0.001), longer duration (P-trend<0.01), and larger proportion of affected pregnancies (P-trend<0.0001) among women with ≥3 pregnancies. Associations were stronger for more recent pregnancies (<5y). Findings did not differ by menopausal status or breast cancer subtype including estrogen receptor and HER2 expression status. Other pregnancy characteristics examined were not associated with risk. We observed strong inverse associations between pregnancy N&V and breast cancer risk. Replication of these findings and exploration of underlying mechanisms could provide important insight into breast cancer etiology and prevention.
PMCID: PMC3737361  PMID: 23737027
gestational hypertension; gestational diabetes; nausea; pre-eclampsia; pregnancy weight gain; vomiting
6.  Adipokines Do Not Mediate the Association of Obesity and Colorectal Adenoma 
Journal of Cancer Epidemiology  2014;2014:371254.
Purpose. The association between obesity and colon neoplasia is well established but the underlying biological mechanisms are not fully understood. Rates of both obesity and colon cancer differ by race. Adipokines have been postulated as contributors to the observed association; however, few studies have examined the mediating effect of adipokines on the obesity-colon adenoma association with consideration of racial differences. Methods. We determined prediagnostic levels of adiponectin and leptin in Caucasians (217 cases and 650 controls) and African Americans (175 cases and 378 controls) participating in the Case Transdisciplinary Research on Energetics and Cancer Colon Adenoma Study. We evaluated mediating effects of adiponectin and leptin on the association of abdominal adiposity and colon adenoma separately according to race using mediational pathway analysis. Results. We observed differences in circulating adipokine concentrations by race; African Americans had higher levels of leptin and lower levels of adiponectin than Caucasians for both adenoma cases and controls (P values <0.001). Leptin and adiponectin did not mediate the waist-to-hip ratio (WHR) adenoma association in either group (all Sobel P values >0.27). Conclusions. We found no evidence that leptin or adiponectin mediates the abdominal obesity-colorectal adenoma pathway. Larger studies on how these associations vary by race, sex, and obesity are needed.
PMCID: PMC4147295  PMID: 25197277
7.  BRCA1 polymorphisms and breast cancer epidemiology in the Western New York Exposures and Breast Cancer (WEB) study 
Genetic epidemiology  2013;37(5):504-511.
Results of studies for the association of BRCA1 genotypes and haplotypes with sporadic breast cancer have been inconsistent. Therefore, a candidate SNP approach was used in a breast cancer case-control study to explore genotypes and haplotypes that have the potential to affect protein functions or levels. In a breast cancer case-control study, genotyping of BRCA1 polymorphisms Q356R, D693N, and E1038G was performed on 1005 cases and 1765 controls. Unconditional, polytomous logistic regression and chi square tests were used to examine the associations of breast cancer with genotypes and haplotypes. Additionally, interactions between genotype and smoking, benign breast disease, family history of breast cancer, BMI, alcohol consumption, and hormonal risk factors, hormone receptor status, and breast cancer pathology were calculated also using logistic regression and chi square. While, sporadic breast cancer was not associated with BRCA1 genotypes or haplotypes overall or by menopausal status, there was evidence of an interaction between the E1038G BRCA1 genotype, smoking, and BMI among premenopausal women (p for interaction=0.01 and 0.045, respectively) and between E1038G and D693N BRCA1 genotypes and hormone therapy use among postmenopausal women (p for interaction=0.01 and 0.02, respectively). There were no other associations found between BRCA1 genotypes and stage, histologic grade, or nuclear grade. However, the D693N SNP was associated with the risk of triple negative breast cancer (OR=2.31 95% CI: 1.08–4.93). The BRCA1 variants studied may play a role in the etiology of triple negative breast cancer and may interact with environmental factors such as hormone therapy or smoking and increase sporadic breast cancer risk.
PMCID: PMC3699175  PMID: 23674270
BRCA1; breast cancer; haplotypes; polymorphisms; gene × environment
8.  The tumor suppressive role of NUMB isoform 1 in esophageal squamous cell carcinoma 
Oncotarget  2014;5(14):5602-5614.
Esophageal quamous cell carcinoma (ESCC) is the predominant histological type of esophageal carcinoma in Asian populations. To date, few biomarkers have been identified for ESCC. In present study, we found a tumor suppressor, NUMB isoform 1 (NUMB-1), as a promising prognostic biomarker for patients with ESCC. NUMB-1 mRNA was downregulated in 66.7% of primary ESCC tissues when compared with matched adjacent non-tumor tissues. The low expression of NUMB-1 was significantly associated with high tumor recurrence (p=0.029) and poor post-operative overall survival (p=0.016). To further explore the underlying mechanisms by which NUMB-1 regulates ESCC, we demonstrated that ectopic expression of NUMB-1 inhibited cell proliferation through inducing G2/M phase arrest, which was accompanied by an increase in p21 and cyclin B1-cdc2 levels. However, it had no impact on apoptosis of ESCC cells. In addition, overexpression of NUMB-1 prevented epithelial-mesenchymal transition, inhibited invasion of ESCC cells and NOTCH pathway, suppressed Aurora-A activity by preventing phosphorylation of Aurora-A at T288 which resulted in cell cycle arrest. Taken together, our findings suggested NUMB-1 functions as a tumor-suppressor and serves as a prognositc biomarker for ESCC patients; thus, NUMB-1 may be a potential novel therapeutic target for treatment of ESCC.
PMCID: PMC4170621  PMID: 24980814
esophageal squamous cell carcinoma; ESCC; NUMB isoform 1; Aurora-A; G2/M arrest
9.  IFATS Collection: Combinatorial Peptides Identify α5β1 Integrin as a Receptor for the Matricellular Protein SPARC on Adipose Stromal Cells 
Stem cells (Dayton, Ohio)  2008;26(10):2735-2745.
The biological features of adipose stromal (stem) cells (ASC), which serve as progenitors for differentiated cells of white adipose tissue (WAT), are still largely undefined. In an initiative to identify functional ASC surface receptors, we screened a combinatorial library for peptide ligands binding to patient-derived ASC. We demonstrate that both primary and cultured human and mouse stromal cells express a conserved receptor targeted by peptides found to mimic SPARC, a matricellular protein that is required for normal WAT development. A signaling receptor for SPARC has not as yet been determined. By using the SPARC-mimicking peptides CMLAGWIPC (termed hPep) and CWLGEWLGC (termed mPep), isolated by panning on human and mouse cells, respectively, we identified the α5β1 integrin complex as a candidate receptor for SPARC. On the basis of these results, we evaluated ASC responses to SPARC or SPARC-mimicking peptide exposure. Our results suggest that extracellular SPARC binds to α5β1 integrin at sites of focal adhesions, an interaction disrupting firm attachment of ASC to extracellular matrix. We propose that SPARC-mediated mobilization of ASC through its effect on α5β1 integrin complex provides a functional basis for the regulation of WAT body composition by SPARC. We also show that α5β1 integrin is a potential target for ASC-selective intracellular delivery of bioactive peptides and gene therapy vectors directed by the SPARC-mimicking peptides.
PMCID: PMC4066418  PMID: 18583538
Adipose stromal cells; Extracellular matrix; Mobilization; Peptide phage display
10.  Low-Dose Decitabine-Based Chemoimmunotherapy for Patients with Refractory Advanced Solid Tumors: A Phase I/II Report 
Journal of Immunology Research  2014;2014:371087.
Aberrant DNA methylation is one of the main drivers of tumor initiation and progression. The reversibility of methylation modulation makes it an attractive target for novel anticancer therapies. Clinical studies have demonstrated that high-dose decitabine, a hypomethylating agent, results in some clinical benefits in patients with refractory advanced tumors; however, they are extremely toxic. Low doses of decitabine minimize toxicity while potentially improving the targeted effects of DNA hypomethylation. Based on these mechanisms, low-dose decitabine combined with chemoimmunotherapy may be a new treatment option for patients with refractory advanced tumors. We proposed the regimen of low-dose decitabine-based chemoimmunotherapy for patients with refractory advanced solid tumors. A favorable adverse event profile was observed in our trial that was highlighted by the finding that most of these adverse events were grades 1-2. Besides, the activity of our cohort was optimistic and the clinical benefit rate was up to 60%, and the median PFS was prolonged compared with PFS to previous treatment. We also identified a significant correlation between the PFS to previous treatment and clinical response. The low-dose DAC decitabine-based chemoimmunotherapy might be a promising protocol for improving the specificity and efficiency of patients with refractory advanced solid tumors. This trial is registered in the database (identifier NCT01799083).
PMCID: PMC4054619  PMID: 24963497
11.  Alcohol intake over the life course and breast cancer survival in Western New York Exposures and Breast Cancer (WEB) study: Quantity and intensity of intake 
Alcohol intake is a risk factor for breast cancer, but the association between alcohol and mortality among breast cancer survivors is poorly understood. We examined the association between alcohol intake from all sources, assessed by cognitive lifetime drinking history, and all-cause and breast cancer mortality among women with breast cancer (N=1097) who participated in a population-based case-control study. Vital status was ascertained through 2006 using the National Death Index. Using Cox Proportional Hazards models, we computed hazard ratios for all-cause and breast cancer mortality in association with alcohol intake. We examined lifetime volume and intensity (drinks per drinking day) of alcohol consumption as well as drinking status during various life periods. Analyses were stratified by menopausal status. After adjustment for total intake, postmenopausal women with consumption of four or more drinks per drinking day over their lifetimes were nearly three times more likely to die from any cause compared to abstainers (HR2.94, 95%CI 1.31,6.62). There was a similar but non-significant association with breast cancer mortality (HR2.68, 95%CI 0.94,7.67). Postmenopausal women who drank one drink or fewer per drinking day between menarche and first birth had a significantly decreased hazard of all-cause (HR0.54, 95%CI 0.31,0.95) and breast cancer mortality (HR0.27, 95%CI 0.09,0.77). Premenopausal breast cancer survival was not associated with drinking intensity. We observed no associations between drinking status or total volume of alcohol intake and breast cancer or all-cause mortality. High-intensity alcohol consumption may be associated with decreased survival in postmenopausal women with breast cancer. Low-intensity alcohol consumption between menarche and first birth may be inversely associated with all-cause and breast cancer mortality; this period may be critical for development of and survival from breast cancer. Intensity of alcohol intake may be a more important factor than absolute volume of intake on survival in women with breast cancer.
PMCID: PMC3667502  PMID: 23605086
breast cancer; alcohol; survival; population-based study
12.  Numb Promotes Cell Proliferation and Correlates with Poor Prognosis in Hepatocellular Carcinoma 
PLoS ONE  2014;9(4):e95849.
Numb is an evolutionary conserved protein that plays critical roles in cell fate determination, cell adhesion, cell migration and a number of signaling pathways, but evidence for a substantial involvement of Numb in HCC has remained unclear. The present study was aimed to investigate the clinical and prognostic significance of Numb and its role in hepatocellular carcinoma (HCC).
The expression of Numb was detected in 107 cases of clinical paraffin-embedded hepatocellular carcinoma tissues,5 matched paris of fresh tissues and six hepatocellular cell lines by immunohistochemistry with clinicopathological analyses,RT-PCR or Western blot. Moreover, loss of function and gain of function assays were performed to evaluate the effect of Numb on cell proliferation in vitro.
We found that Numb was obviously up-regulated in HCC tissues and cell lines (p<0.05). The Numb up-regulation correlated significantly with poor prognosis, and Numb status was identified as an independent prognostic factor. Over-expression of Numb increased proliferation in SMMC-7721 and BEL-7402 cells, while knock-down of Numb showed the opposite effect. Our study indicates that Numb up-regulation significantly correlates with cell proliferation and poor prognosis in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy in hepatocellular carcinoma treatment.
PMCID: PMC4000222  PMID: 24770339
14.  Intestinal obstruction due to migration of a thermometer from bladder to abdominal cavity: A case report 
Intraperitoneal foreign bodies such as retained surgical instruments can cause intestinal obstruction. However, intestinal obstruction due to transmural migration of foreign bodies has rarely been reported. Here, we report a case of intestinal obstruction due to a clinical thermometer which migrated from the bladder into the abdominal cavity. A 45-year-old man was admitted to our hospital with a one-year history of recurrent lower abdominal cramps. Two days before admission, the abdominal cramps aggravated. Intestinal obstruction was confirmed with upright abdominal radiography and computerized tomography scan which showed dilation of the small intestines and a thermometer in the abdominal cavity. Then laparotomy was performed. A scar was observed at the fundus of the bladder and a thermometer was adhering to the small bowels and mesentery which resulted in intestinal obstruction. Abdominal cramps were eliminated and defecation and flatus recovered soon after removal of the thermometer.
PMCID: PMC3942848  PMID: 24605042
Intestinal obstruction; Foreign body; Thermometer; Transmural migration; Bladder
15.  Exposure to traffic emissions: associations with biomarkers of antioxidant status and oxidative damage 
Environmental research  2012;121:31-38.
Oxidative stress has been implicated as a possible mechanism for adverse health effects associated with traffic emissions. We examined the association of an estimate of traffic emissions with blood biomarkers of antioxidant capacity (glutathione, glutathione peroxidase, trolox-equivalent antioxidant capacity) and oxidative damage (thiobarbituric acid-reactive substances (TBARS)) among 1,810 healthy women, randomly selected from Erie and Niagara Counties in Western New York.
A geographic traffic emission and meteorological dispersion model was used to estimate annual polycyclic aromatic hydrocarbon (PAH) exposure from traffic emissions for each woman based on her residence at the time of study. Associations of traffic-related PAH exposure with measures of oxidative stress and antioxidant capacity were examined in multiple regression analyses with adjustment for potential confounders.
Higher traffic-related PAH exposure was associated with decreased glutathione and increased glutathione peroxidase. Stronger associations between traffic-related PAH exposure and levels of glutathione and glutathione peroxidase were suggested among nonsmoking women without secondhand smoke exposure, especially among premenopausal nonsmoking women. Associations were also stronger for measurements made in warmer months.
These findings suggest that PAHs or other components of traffic emissions may impact anti-oxidative capacity among healthy women, particularly premenopausal non-smokers without secondhand smoke exposure.
PMCID: PMC3578064  PMID: 23140610
Traffic emissions; Polycyclic aromatic hydrocarbon; Oxidative stress
16.  Single nucleotide polymorphisms in uracil-processing genes, intake of one-carbon nutrients and breast cancer risk 
The misincorporation of uracil into DNA leads to genomic instability. In a prior study, some of us identified four common SNPs in uracil-processing genes (rs2029166 and rs7296239 in SMUG1, rs34259 in UNG, and rs4775748 in DUT) that were associated with significantly altered levels of uracil in human DNA. We investigated whether any of these SNPs are associated with an altered risk of developing breast cancer and if one-carbon nutrients intake can modify their effects.
We genotyped the four SNPs in 1,077 cases of incident breast cancer and 1,910 age and race-matched controls in the Western New York Exposures and Breast Cancer (WEB) Study and examined associations with breast cancer risk and interactions with intake of folate, vitamins B6 and B12.
After adjustment for known risk factors for breast cancer, there was increased risk of breast cancer among postmenopausal women who were heterozygous for either of the SMUG1 SNPs (OR 1.29, 95% CI 1.07–1.56) and (1.29, 1.07–1.55). Among premenopausal women, increased risk associated with the SMUG1 rs2029166 genotype was limited to those with low folate intake. There were no other interactions with vitamins B6 or B12 intake.
Our study suggests that the four selected SNPs are not robust determinants of breast cancer risk, but that the two SNPs in SMUG1 might modestly alter the risk of breast cancer. However, the increase in risk among heterozygotes in the two SNPs in SMUG1, which is thought to be the most active glycosylase in vivo, raises the possibility that subtle ‘heterosis’ effects on cancer risk might be produced by these SNPs.
PMCID: PMC3902360  PMID: 21427733
Uracil-Processing Genes; Single nucleotide polymorphisms; Breast cancer
17.  Advanced Oxidation Protein Products Activate Intrarenal Renin–Angiotensin System via a CD36-Mediated, Redox-Dependent Pathway 
Antioxidants & Redox Signaling  2013;18(1):19-35.
Aims: Activation of intrarenal renin–angiotensin system (RAS) has a detrimental effect on the progression of chronic kidney diseases (CKDs), although the regulation of intrarenal RAS remains unclear. The aim of the present study was to evaluate the role of advanced oxidation protein products (AOPPs) in intrarenal RAS activation. Results: AOPPs upregulated the expression of almost all components of RAS and increased activity of angiotensin-converting enzyme in cultured proximal tubular epithelial cells. The triggering effect of AOPP-albumin was 100-times stronger than that of unmodified albumin. The effect of AOPP-albumin was mainly mediated by a CD36-dependent, redox-sensitive signaling involving activation of protein kinase Cα, NADPH oxidase, and nuclear factor-κB/activation protein-1. Chronic AOPP–albumin loading in unilateral nephrectomy rats resulted in deposition of AOPPs in renal tubular cells accompanied with local RAS activation and functional perturbations such as increase in urinary albumin excretion. Accumulation of AOPPs was also detected in human renal tubular cells and correlated with expression of angiotensin II in renal biopsies from 19 patients with IgA nephropathy. Innovation and Conclusion: This study demonstrated for the first time that AOPPs modified albumin functions as a strong trigger of intrarenal RAS via a CD36-mediated, redox-dependent pathway. Given the fact that accumulation of AOPPs is prevalent in diabetes and CKD, targeting AOPPs could be a strategy for the therapeutic intervention of CKD. Antioxid. Redox Signal. 18, 19–35.
PMCID: PMC3503474  PMID: 22662869
18.  DNA promoter methylation in breast tumors: No association with genetic polymorphisms in MTHFR and MTR 
Aberrant promoter methylation is recognized as an important feature of breast carcinogenesis. We hypothesized that genetic variation of genes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR), two critical enzymes in one-carbon metabolism, may alter DNA methylation levels, and thus influence DNA methylation in breast cancer. We evaluated case-control association of MTHFR C677T, A1298C, and MTR A2756G polymorphisms for cases strata defined by promoter methylation status for each of three genes, E- cadherin, p16, and RAR-β2 in breast cancer; in addition, we evaluated case-case comparisons of likelihood of promoter methylation in relation to genotypes using a population-based case-control study conducted in Western New York State. Methylation was evaluated with real time methylation-specific PCRs for 803 paraffin embedded breast tumor tissues from women with primary, incident breast cancer. We applied unordered polytomous regression and unconditional logistic regression to derive adjusted odds ratios (OR) and 95% confidence intervals (CI). We did not find any association of MTHFR and MTR polymorphisms with breast cancer risk stratified by methylation status nor between polymorphisms and likelihood of promoter methylation of any of the genes. There was no evidence of difference within strata defined by menopausal status, ER status, folate intake and lifetime alcohol consumption. Overall, we found no evidence that these common polymorphisms of the MTHFR and MTR genes are associated with promoter methylation of E- cadherin, p16, and RAR-β2 genes in breast cancer.
PMCID: PMC3837294  PMID: 19240236
promoter methylation; MTHFR; MTR; breast cancer; epidemiology; genetic polymorphisms
19.  No Association between Circulating Levels and Genetic Variants of IL-6 and TNF-α and Colon Adenoma 
Gastroenterology research  2013;6(2):10.4021/gr529w.
Background & Aims
Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), two important inflammatory cytokines, have been inconsistently associated with risk of colon neoplasia in epidemiological studies. However, research to date has not adequately assessed whether race-specific differences may exist in associations between biomarkers and genetic variants of these cytokines and colorectal adenoma – the precursor lesions of colorectal cancer. We sought to determine whether circulating levels of IL-6 and TNF-α, or genetic polymorphisms in IL-6and TNF-α were associated with colon adenoma and if so, whether that association differed by race.
We analyzed the associations of circulating levels and single nucleotide polymorphisms (SNPs) of IL-6 and TNF-α with risk of colon adenomas in a colonoscopy -based case-control study of 401 incident adenoma cases and 1,050 controls. We used multivariate unconditional logistic regression models to estimate the odds ratios (OR) and 95% confidence intervals (95% CI) for levels or genotypes (log additive models) of IL-6 and TNF-α.
Compared to the bottom tertile of IL-6, the adjusted ORs were: 1.06 (0.75–1.44), 1.01 (0.72–1.40), respectively for the 2nd and 3rd tertiles (ptrend = 0.10); the corresponding ORs for TNF-α were: 0.85 (0.63–1.15) and 1.01 (0.75–1.36), respectively (ptrend = 0.39). Race-stratified analyses did not reveal any significant association. There were also no statistically significant associations between IL-6 and TNF-α SNPs and colon adenoma.
Our results do not support pre-diagnostic levels of IL-6, TNF-α or their genetic variants as significant risk factors for the development of colon adenoma.
PMCID: PMC3825207  PMID: 24235998
20.  A Network Pharmacology Approach to Evaluating the Efficacy of Chinese Medicine Using Genome-Wide Transcriptional Expression Data 
The research of multicomponent drugs, such as in Chinese Medicine, on both mechanism dissection and drug discovery is challenging, especially the approaches to systematically evaluating the efficacy at a molecular level. Here, we presented a network pharmacology-based approach to evaluating the efficacy of multicomponent drugs by genome-wide transcriptional expression data and applied it to Shenmai injection (SHENMAI), a widely used Chinese Medicine composed of red ginseng (RG) and Radix Ophiopogonis (RO) in clinically treating myocardial ischemia (MI) diseases. The disease network, MI network in this case, was constructed by combining the protein-protein interactions (PPI) involved in the MI enriched pathways. The therapeutic efficacy of SHENMAI, RG, and RO was therefore evaluated by a network parameter, namely, network recovery index (NRI), which quantitatively evaluates the overall recovery rate in MI network. The NRI of SHENMAI, RG, and RO were 0.876, 0.494, and 0.269 respectively, which indicated SHENMAI exerts protective effects and the synergistic effect of RG and RO on treating myocardial ischemia disease. The successful application of SHENMAI implied that the proposed network pharmacology-based approach could help researchers to better evaluate a multicomponent drug on a systematic and molecular level.
PMCID: PMC3666440  PMID: 23737854
21.  The Oncogenic Role of microRNA-130a/301a/454 in Human Colorectal Cancer via Targeting Smad4 Expression 
PLoS ONE  2013;8(2):e55532.
Transforming growth factor (TGF)-β/Smad signaling plays an important role in colon cancer development, progression and metastasis. In this study we demonstrated that the microRNA-130a/301a/454 family is up-regulated in colon cancer tissues compared to paired adjacent normal mucosa, which share the same 3′-untranslational region (3′-UTR) binding seed sequence and are predicated to target Smad4. In colorectal cancer HCT116 and SW480 cells, overexpression of miRNA-130a/301a/454 mimics enhances cell proliferation and migration, while inhibitors of these miRNAs affect cell survival. The biological function of miRNA-130a/301a/454 on colon cancer cells is likely mediated by suppression of Smad4, and the up-regulation of the miRNAs is correlated with Smad4 down-regulation in human colon cancers. Collectively, these results suggest that miRNA-130a/301a/454 are novel oncogenic miRNAs contributing to colon tumorigenesis by regulating TGF-β/Smad signaling, which may have potential application in cancer therapy.
PMCID: PMC3564758  PMID: 23393589
22.  Use of non-steroidal anti-inflammatory drugs and survival following breast cancer diagnosis 
While there is accumulating evidence that use of non-steroidal anti-inflammatory drugs (NSAIDs) decreases breast cancer risk, little is known about the impact of NSAIDs on survival after breast cancer diagnosis.
We assessed whether recent, pre-diagnostic NSAID use and lifetime cumulative aspirin use before diagnosis were associated with survival among 1,024 women with incident, primary, invasive breast cancer.
Recent, pre-diagnostic use of aspirin, ibuprofen, and acetaminophen, and lifetime use of aspirin up to diagnosis were not associated with either all-cause mortality or breast cancer specific mortality. Neither dose nor frequency of use was associated with risk. Associations were not different for pre- and post-menopausal women.
In our data, pre-diagnostic NSAID use and lifetime cumulative aspirin use were not associated with breast cancer survival.
Our findings do not support a role of NSAIDs prior to diagnosis in breast cancer survival.
PMCID: PMC3253909  PMID: 22068285
23.  Intrachromosomal Changes and Genomic Instability in Site-Specific Microbeam-Irradiated and Bystander Human-Hamster Hybrid Cells 
Radiation research  2011;177(1):25-34.
Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (AL) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.
PMCID: PMC3410536  PMID: 22077336
24.  AGE-LDL Activates Toll Like Receptor 4 Pathway and Promotes Inflammatory Cytokines Production in Renal Tubular Epithelial Cells 
Background/Aims:Accumulation of advanced glycation end-products, the well-recognized pro-inflammatory molecules, has been detected in renal tissues including tubules. The aim of the present study was to investigate the role of advanced glycation end-products modified low density lipoprotein (AGE-LDL) in inflammatory cytokines production in human proximal tubular epithelial cells and the underlying mechanism. Methods: The Interleukin-6 (IL-6) and Interleukin-8 (IL-8) production was examined by real-time PCR and ELISA. The expression of Toll-like receptor 2 and 4 (TLR2/4) was detected by flow cytometry and western blot. The interaction of TLR2/4 with AGE-LDL was examined by co-immunoprecipitation assay. The involvement of MyD88 and the downstream molecules in inflammatory cytokines production was examined by siRNA and pharmacologic inhibitors, respectively. Results: AGE-LDL interacted with TLR2 and TLR4. TLR4 siRNA showed stronger inhibition on AGE-LDL-induced IL-6 and IL-8 production than that of TLR2 siRNA. Silencing MyD88, but not TRIF, inhibited AGE-LDL-induced IL-6 and IL-8 production. AGE-LDL stimulation led to phosphorylation of JNK, p38, Akt and the p65 subunit of nuclear factor-κB (NF-κB). Pharmacologic inhibitor of Akt suppressed AGE-LDL-induced activation of NF-κB, but the inhibitor of JNK, p38 or ERK1/2 had no effect. Blocking MyD88, p38, JNK, Akt or NF-κB attenuated AGE-LDL-triggered IL-6 production. Conclusion: AGE-LDL induced IL-6 and IL-8 production via TLR2/4-MyD88-dependent pathway in tubular epithelial cells. These data suggest that activation of TLRs signaling in tubular epithelial cells by AGE-LDL might be a novel mechanism for the tubulointerstitial inflammation.
PMCID: PMC3555149  PMID: 23355795
AGE-LDL; Toll like receptor; MyD88; NF-κB; IL-6.
25.  An atlas of DNA methylomes in porcine adipose and muscle tissues 
Nature communications  2012;3:850.
It is evident that epigenetic factors, especially DNA methylation, play essential roles in obesity development. Using pig as a model, here we investigated the systematic association between DNA methylation and obesity. We sampled eight variant adipose and two distinct skeletal muscle tissues from three pig breeds living within comparable environments but displaying distinct fat level. We generated 1,381 gigabases (Gb) of sequence data from 180 methylated DNA immunoprecipitation (MeDIP) libraries, and provided a genome-wide DNA methylation map as well as a gene expression map for adipose and muscle studies. The analysis showed global similarity and difference among breeds, sexes and anatomic locations, and identified the differentially methylated regions (DMRs). The DMRs in promoters are highly associated with obesity development via expression repression of both known obesity-related genes and novel genes. This comprehensive map provides a solid basis for exploring epigenetic mechanisms of adipose deposition and muscle growth.
PMCID: PMC3508711  PMID: 22617290

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