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author:("Zhang, shuman")
1.  Community dynamics of prokaryotic and eukaryotic microbes in an estuary reservoir 
Scientific Reports  2014;4:6966.
This study demonstrates both prokaryotic and eukaryotic community structures and dominant taxonomies in different positions of the greatest estuary reservoir for drinking water source in the world in four seasons of one year using 454 pyrosequencing method with total of 312,949 16S rRNA and 374,752 18S rRNA gene fragments, including 1,652 bacteria OTUs and 1,182 fungus OTUs. During winter and spring, the community composition at the phylum level showed that microorganisms had similar structures but their quantities were different. Similarly, obvious changes at the genus level were observed among the samples taken in winter and spring between summer and fall. Microorganisms located the reservoir inlet were founded to be different from those in rear at both phylum and genus level. Air temperature had a stronger effect than sampling location on the microbial community structure. Total nitrogen and dissolved oxygen were algae-monitoring indicators during the whole year. Moreover, Bacillus was an efficient indicator during summer and autumn for bacteria OTUs.
doi:10.1038/srep06966
PMCID: PMC4225533  PMID: 25382138
2.  MK-2206, an Akt inhibitor, enhances carboplatinum/paclitaxel efficacy in gastric cancer cell lines 
Cancer Biology & Therapy  2013;14(10):932-936.
Background
Several molecularly-targeted agents are being evaluated in gastric cancer cell lines. In this study we evaluated the synergistic potential of MK-2206, an oral potent allosteric Akt inhibitor, in combination with chemotherapeutic agents in human gastric cancer cell lines.
Materials and Methods
We evaluated effects of MK-2206 on cell growth and cell signaling using a panel of gastric cancer cell lines AGS, SNU-1 and SNU 16. The analysis of drug combinations was conducted by using CellTiter-Blue™ Cell Viability Assay which yielded the combination index (CI). MK-2206 and representative chemotherapy agent were further evaluated regarding their effects on Akt inhibition and downstream targets using western blots probed with the appropriate antibodies. We assessed the combination of MK-2206 and chemotherapy in three different treatment sequences.
Results
We demonstrated in vitro synergistic efficacy of MK-2206 when combined with carboplatinum and paclitaxel in the three cell lines examined. Efficacy was dose dependent. We assessed the combination of MK-2206 and carboplatinum/paclitaxel in three different treatment sequences; 24 h of exposure to combination chemotherapy followed by a 48 h exposure to MK-2206 resulted in the highest synergistic antiproliferative effect in all cell lines. On the other hand, the reverse sequence (MK-2206 followed by chemotherapy) and the concurrent treatment schedule were slightly synergistic or additive as well. The effects of MK-2206 on p-Akt and other downstream targets was reported.
Conclusions
Our findings suggest that Akt inhibition augments the efficacy of existing gastric cancer therapeutics (carboplatinum and paclitaxel); thus, MK-2206 is a promising agent to treat gastric cancer patients who receive these cytotoxic agents. The magnitude of synergy depended on the treatment sequence; a schedule of MK-2206 dosed before or concurrently with chemotherapy was not as effective as being dosed after chemotherapy. Further experiments addressing MK-2206’s mechanism of action in combination with chemotherapy are needed.
doi:10.4161/cbt.25939
PMCID: PMC3926890  PMID: 23917345
MK-2206; carboplatinum; paclitaxel; gastric cancer
3.  Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice 
Hepatology (Baltimore, Md.)  2013;58(2):603-616.
Obesity is a calorie excessive state that is associated with high risk of diabetes, atherosclerosis and certain types of tumors. Obesity may induce inflammation and insulin resistance. We found that the expression of interferon regulatory factor 9 (IRF9), a major transcription factor mediating interferon (IFN) responses, was lower in the livers of obese mice than in those of their lean counterparts. Furthermore, whole-body IRF9 knockout (KO) mice were more obese and had aggravated insulin resistance, hepatic steatosis and inflammation after chronic high-fat diet (HFD) feeding. In contrast, adenoviral-mediated hepatic IRF9 overexpression in both diet-induced and genetically (ob/ob) obese mice showed markedly improved hepatic insulin sensitivity and attenuated hepatic steatosis and inflammation. We further employed a yeast two-hybrid screening system to investigate the interactions between IRF9 and its cofactors. Importantly, we identified that IRF9 interacts with peroxisome proliferator-activated receptor α (PPARα), an important metabolism-associated nuclear receptor, to activate PPARα target genes. In addition, liver-specific PPARα overexpression rescued insulin sensitivity and ameliorated hepatic steatosis and inflammation in IRF9 KO mice. Taken together, our results indicate that IRF9 attenuates hepatic insulin resistance, steatosis and inflammation through interaction with PPARα
doi:10.1002/hep.26368
PMCID: PMC3736732  PMID: 23471885
high-fat diet; diabetes; metabolism; inflammation; PPARα
4.  A prospective analysis of body size during childhood, adolescence, and adulthood and risk of non-Hodgkin lymphoma 
The etiology of non-Hodgkin lymphoma (NHL) is poorly understood. Obesity is associated with inflammation, a cytokine milieu conducive to lymphocyte proliferation, and has been associated with NHL risk in some epidemiologic studies. To prospectively examine NHL risk in relation to adult and earlier life obesity, we documented 635 incident NHL diagnoses among 46,390 men in the Health Professionals Follow-up Study and 1254 diagnoses among 116,794 women in the Nurses’ Health Study over 22–32 years of follow-up. Using multivariable Cox proportional hazards models we estimated cohort-specific incidence rate ratios (RRs) and 95% confidence intervals (CI) for risk of NHL and major histologic subtypes associated with cumulative average middle and young adult (ages 18–21) body mass index (BMI) and adolescent and childhood somatotype. NHL risk was modestly increased in men (but not women) with a cumulative average middle adult BMI ≥30 kg/m2 (vs. 15–22.9 kg/m2; RR: 1.28; 95% CI: 0.92, 1.77; P-trend=0.05). In meta-analyses across cohorts, higher young adult BMI was associated with increased risk of all NHL (pooled RR per 5 kg/m2: 1.19; 95% CI: 1.05, 1.37), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) (all P-trend≤0.02). Adolescent somatotype was also positively associated with all NHL, DLBCL, and FL in pooled analyses (all P-trend ≤0.03) while childhood somatotype was positively associated with NHL overall among women only (P-trend <0.01). These findings in two large prospective cohorts provide novel evidence that larger body size in childhood, adolescence, and young adulthood predicts increased risk of NHL, and particularly of DLBCL and FL.
doi:10.1158/1940-6207.CAPR-13-0132
PMCID: PMC3761937  PMID: 23803416
non-Hodgkin lymphoma; obesity; body mass index; anthropometry; epidemiology
5.  Alternate-Day Low-Dose Aspirin and Cancer Risk: Long-term Observational Follow-up of a Randomized Trial 
Annals of internal medicine  2013;159(2):77-85.
Background
Observational studies and meta-analyses of trials suggest daily aspirin use may affect cancer risk, particularly for colorectal cancer, but evidence regarding alternate-day use is scant.
Objective
To examine the association between long-term use of alternate-day low-dose aspirin and cancer incidence in healthy women.
Design
Observational follow-up of a randomized controlled trial.
Setting
U.S. female health professionals.
Participants
39,876 women aged 45 and over in the Women’s Health Study, 33,682 of whom continued observational follow-up.
Intervention
100 mg of aspirin or placebo administered every other day until March 2004, with a median 10-year follow-up. Post-trial observational follow-up continued through March 2012.
Measurements
Incidence of cancer.
Results
5,071 cancers were confirmed throughout follow-up, including 2,070 breast, 451 colorectal, 431 lung cancers, and 1,391 cancer deaths. Over the entire follow-up there was no overall effect of aspirin on total (hazard ratio (HR) = 0.97, 95% confidence interval (CI) = 0.92-1.03, p=0.31), breast (HR=0.98, 95% CI = 0.90-1.07 p=0.65) or lung (HR=1.04, 95% CI = 0.86-1.26, p=0.67) cancer. Incidence of colorectal cancer was lower in the aspirin group (HR=0.80, 95% CI = 0.67-0.97, p=0.021), primarily due to a reduction in proximal colon cancer (HR=0.73, 95% CI = 0.55-0.95, p=0.022), with the effect emerging after 10 years. The post-trial reduction in colorectal cancer was 42% (HR=0.58, 95% CI = 0.42-0.80, p<0.001). There was no extended effect on cancer deaths or colorectal polyps. There were more reported gastrointestinal bleeds (HR=1.14, 95% CI=1.06-1.22, p<0.001) and peptic ulcers (HR=1.17, 95% CI=1.09-1.27, p<0.001) in the aspirin group.
Limitations
Data were available only for women. Not all women received extended follow-up, and the possibility of ascertainment bias post-trial cannot be ruled out. Gastrointestinal bleeding, peptic ulcer, and polyp information was obtained only from self-report during extended follow-up.
Conclusions
Long-term use of alternate-day, low-dose aspirin may reduce risk for colorectal cancer in healthy women.
doi:10.7326/0003-4819-159-2-201307160-00002
PMCID: PMC3713531  PMID: 23856681
6.  Global Population Structure and Evolution of Bordetella pertussis and Their Relationship with Vaccination 
mBio  2014;5(2):e01074-14.
ABSTRACT
Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines.
IMPORTANCE
Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.
doi:10.1128/mBio.01074-14
PMCID: PMC3994516  PMID: 24757216
7.  Association between Sex Hormones and Colorectal Cancer Risk in Men and Women 
Background & Aims
There is observational and clinical evidence that indicate that sex hormones affect development of colorectal cancer (CRC) in men and women. However, the relationship between endogenous sex hormone levels and CRC is unclear.
Methods
We collected data on lifestyle, medical history, and diet etc. (through 2008), along with blood samples, from the Nurses’ Health Study, the Women’s Health Study, the Health Professional Follow-Up Study, and the Physicians’ Health Study II. We measured plasma levels of estrone, estradiol, testosterone, sex hormone binding globulin (SHBG), and c-peptide among 730 women (293 cases of CRC and 437 healthy individuals, as controls) and 1158 men (439 CRC cases and 719 controls), and used unconditional logistic regression to estimate relative risks (RRs) and 95% confidence intervals (CIs). All statistical tests were 2-sided.
Results
Total testosterone, SHBG, and the ratio of estradiol to testosterone were associated with CRC in men after adjustments for matching and risk factors for CRC, including BMI and plasma levels of C-peptide. The RRs in the highest relative to the lowest quartile were 0.62 for testosterone (95% CI, 0.40–0.96), 0.65 for SHBG (95% CI, 0.42−0.99), and 2.63 for the ratio (95% CI, 1.58–4.36) (P-values for trend ≤0.02). However, in women, only the ratio of estradiol to testosterone was (inversely) associated with CRC after adjustments for all factors (RR, 0.43; 95% CI, 0.22−0.84; P-value for trend, .03).
Conclusions
Based on combined data from 4 population studies, there appears to be an association between levels of sex hormones and CRC risk in men. There also appears to be an inverse association between the ratio of estradiol to testosterone and CRC in postmenopausal women.
doi:10.1016/j.cgh.2012.11.012
PMCID: PMC3594467  PMID: 23200979
estrogen; incidence; colorectal cancer; testosterone
8.  Cathelicidins from the Bullfrog Rana catesbeiana Provides Novel Template for Peptide Antibiotic Design 
PLoS ONE  2014;9(3):e93216.
Cathelicidins, a class of gene-encoded effector molecules of vertebrate innate immunity, provide a first line of defense against microbial invasions. Although cathelicidins from mammals, birds, reptiles and fishes have been extensively studied, little is known about cathelicidins from amphibians. Here we report the identification and characterization of two cathelicidins (cathelicidin-RC1 and cathelicidin-RC2) from the bullfrog Rana catesbeiana. The cDNA sequences (677 and 700 bp, respectively) encoding the two peptides were successfully cloned from the constructed lung cDNA library of R. catesbeiana. And the deduced mature peptides are composed of 28 and 33 residues, respectively. Structural analysis indicated that cathelicidin-RC1 mainly assumes an amphipathic alpha-helical conformation, while cathelicidin-RC2 could not form stable amphipathic structure. Antimicrobial and bacterial killing kinetic analysis indicated that the synthetic cathelicidin-RC1 possesses potent, broad-spectrum and rapid antimicrobial potency, while cathelicidin-RC2 exhibited very weak antimicrobial activity. Besides, the antimicrobial activity of cathelicidin-RC1 is salt-independent and highly stable. Scanning electron microscopy (SEM) analysis indicated that cathelicidin-RC1 kills microorganisms through the disruption of microbial membrane. Moreover, cathelicidin-RC1 exhibited low cytotoxic activity against mammalian normal or tumor cell lines, and low hemolytic activity against human erythrocytes. The potent, broad-spectrum and rapid antimicrobial activity combined with the salt-independence, high stability, low cytotoxic and hemolytic activities make cathelicidin-RC1 an ideal template for the development of novel peptide antibiotics.
doi:10.1371/journal.pone.0093216
PMCID: PMC3968123  PMID: 24675879
9.  Fruit and Vegetable Intake and Risk of Breast Cancer by Hormone Receptor Status 
Background
Estrogen receptor–negative (ER−) breast cancer has few known or modifiable risk factors. Because ER− tumors account for only 15% to 20% of breast cancers, large pooled analyses are necessary to evaluate precisely the suspected inverse association between fruit and vegetable intake and risk of ER− breast cancer.
Methods
Among 993 466 women followed for 11 to 20 years in 20 cohort studies, we documented 19 869 estrogen receptor positive (ER+) and 4821 ER− breast cancers. We calculated study-specific multivariable relative risks (RRs) and 95% confidence intervals (CIs) using Cox proportional hazards regression analyses and then combined them using a random-effects model. All statistical tests were two-sided.
Results
Total fruit and vegetable intake was statistically significantly inversely associated with risk of ER− breast cancer but not with risk of breast cancer overall or of ER+ tumors. The inverse association for ER− tumors was observed primarily for vegetable consumption. The pooled relative risks comparing the highest vs lowest quintile of total vegetable consumption were 0.82 (95% CI = 0.74 to 0.90) for ER− breast cancer and 1.04 (95% CI = 0.97 to 1.11) for ER+ breast cancer (P common-effects by ER status < .001). Total fruit consumption was non-statistically significantly associated with risk of ER− breast cancer (pooled multivariable RR comparing the highest vs lowest quintile = 0.94, 95% CI = 0.85 to 1.04).
Conclusions
We observed no association between total fruit and vegetable intake and risk of overall breast cancer. However, vegetable consumption was inversely associated with risk of ER− breast cancer in our large pooled analyses.
doi:10.1093/jnci/djs635
PMCID: PMC3593764  PMID: 23349252
10.  Confocal microscopy study of pertussis toxin and toxoids on CHO-cells 
Pertussis toxin in its detoxified form is a major component of all current acellular pertussis vaccines. Here we report the membrane translocation and internalization activities of pertussis toxin and various pertussis toxoids using Chinese hamster ovary cells and confocal microscopy based on indirect immunofluorescence labeling. Chemically detoxified pertussis toxoids were able to translocate/internalize into cells at the concentration about 1,000 times higher than the native toxin. Pertussis toxoids detoxified with different procedures (glutaraldehyde, glutaraldehyde plus formaldehyde, hydrogen peroxide or genetic mutation) showed differences in fluorescence intensity under the same condition, indicating toxoids from different detoxification methods may have different translocation/internalization activities on cells.
doi:10.4161/hv.22795
PMCID: PMC3859756  PMID: 23291938
CHO cells; confocal microscopy; detoxification; pertussis toxin; toxoid; translocation
11.  Effect of Combined Folic Acid, Vitamin B6, and Vitamin B12 on Colorectal Adenoma 
Background
Folic acid, vitamin B6, and vitamin B12 act in concert in the one-carbon metabolism and may protect against colorectal neoplasia. We examined the effect of combined B-vitamin treatment on the occurrence of colorectal adenoma.
Methods
The Women’s Antioxidant and Folic Acid Cardiovascular Study was a randomized, double-blind, placebo-controlled trial of 5442 female health professionals at high risk for cardiovascular disease from April 1998 through July 2005. Participants were randomly assigned to receive a combination pill of folic acid (2.5mg), vitamin B6 (50mg), and vitamin B12 (1mg) or placebo. This study included 1470 participants who were followed up for as long as 9.2 years and underwent an endoscopy at any point during follow-up. We estimated relative risks using a generalized linear model with a natural logarithm link function and Poisson distributed errors. All statistical tests were two-sided.
Results
The risk of colorectal adenoma was similar among participants receiving treatment (24.3%, 180 of 741 participants) vs placebo (24.0%, 175 of 729 participants) (multivariable adjusted relative risk = 1.00, 95% confidence interval = 0.83 to 1.20). Treatment was not associated with the risk of adenoma when data were analyzed by subsite, size, stage, and the number of adenomas. There was no statistically significant effect modification by alcohol intake, history of cancer or adenoma, or baseline plasma levels or intakes of folate, vitamin B6, or vitamin B12.
Conclusion
Our results indicate no statistically significant effect of combined folic acid, vitamin B6, and vitamin B12 treatment on colorectal adenoma among women at high risk for cardiovascular disease.
doi:10.1093/jnci/djs370
PMCID: PMC3611818  PMID: 23066166
12.  Prediction of breast cancer risk by genetic risk factors, overall and by hormone receptor status 
Journal of medical genetics  2012;49(9):601-608.
Objective
There is increasing interest in adding common genetic variants identified through genome wide association studies (GWAS) to breast cancer risk prediction models. First results from such models showed modest benefits in terms of risk discrimination. Heterogeneity of breast cancer as defined by hormone-receptor status has not been considered in this context. In this study we investigated the predictive capacity of 32 GWAS-detected common variants for breast cancer risk, alone and in combination with classical risk factors, and for tumors with different hormone receptor status.
Material and Methods
Within the Breast and Prostate Cancer Cohort Consortium (BPC3), we analyzed 6009 invasive breast cancer cases and 7827 matched controls of European ancestry, with data on classical breast cancer risk factors and 32 common gene variants identified through GWAS. Discriminatory ability with respect to breast cancer of specific hormone receptor-status was assessed with the age- and cohort-adjusted concordance statistic (AUROCa). Absolute risk scores were calculated with external reference data. Integrated discrimination improvement (IDI) was used to measure improvements in risk prediction.
Results
We found a small but steady increase in discriminatory ability with increasing numbers of genetic variants included in the model (difference in AUROCa going from 2.7 to 4%). Discriminatory ability for all models varied strongly by hormone receptor status
Discussion and Conclusion
Adding information on common polymorphisms provides small but statistically significant improvements in the quality of breast cancer risk prediction models. We consistently observed better performance for receptor positive cases, but the gain in discriminatory quality is not sufficient for clinical application.
doi:10.1136/jmedgenet-2011-100716
PMCID: PMC3793888  PMID: 22972951
breast cancer; risk prediction; genetic factors; hormone receptor status
13.  A rare collision tumor of squamous carcinoma and small cell carcinoma in esophagus involved with separate lymph nodes: a case report 
Journal of Thoracic Disease  2013;5(5):E203-E206.
We report a case of an esophageal collision tumor composed of squamous cell carcinoma and small cell carcinoma (SmCC). A 66-year-old man complained of chest pain after oral intake for nearly one month. The patient received two cycles of neoadjuvant platinum-based combination chemotherapy and enhanced computed tomography showed a partial response of the tumor. He then underwent a thoracolaparoscopic esophagectomy with extensive mediastinal lymphadenectomy. Two cycles of chemotherapy and prophylactic irradiation of the lymphatic drainage region were sequentially achieved after surgery. The patient has survived for more than 18 months with no evidence of recurrent disease since surgical resection.
doi:10.3978/j.issn.2072-1439.2013.09.13
PMCID: PMC3815725  PMID: 24255793
Esophageal cancer; collision tumor; small cell carcinoma; squamous cell carcinoma
14.  Magnesium, vitamin D status and mortality: results from US National Health and Nutrition Examination Survey (NHANES) 2001 to 2006 and NHANES III 
BMC Medicine  2013;11:187.
Background
Magnesium plays an essential role in the synthesis and metabolism of vitamin D and magnesium supplementation substantially reversed the resistance to vitamin D treatment in patients with magnesium-dependent vitamin-D-resistant rickets. We hypothesized that dietary magnesium alone, particularly its interaction with vitamin D intake, contributes to serum 25-hydroxyvitamin D (25(OH)D) levels, and the associations between serum 25(OH)D and risk of mortality may be modified by magnesium intake level.
Methods
We tested these novel hypotheses utilizing data from the National Health and Nutrition Examination Survey (NHANES) 2001 to 2006, a population-based cross-sectional study, and the NHANES III cohort, a population-based cohort study. Serum 25(OH)D was used to define vitamin D status. Mortality outcomes in the NHANES III cohort were determined by using probabilistic linkage with the National Death Index (NDI).
Results
High intake of total, dietary or supplemental magnesium was independently associated with significantly reduced risks of vitamin D deficiency and insufficiency respectively. Intake of magnesium significantly interacted with intake of vitamin D in relation to risk of both vitamin D deficiency and insufficiency. Additionally, the inverse association between total magnesium intake and vitamin D insufficiency primarily appeared among populations at high risk of vitamin D insufficiency. Furthermore, the associations of serum 25(OH)D with mortality, particularly due to cardiovascular disease (CVD) and colorectal cancer, were modified by magnesium intake, and the inverse associations were primarily present among those with magnesium intake above the median.
Conclusions
Our preliminary findings indicate it is possible that magnesium intake alone or its interaction with vitamin D intake may contribute to vitamin D status. The associations between serum 25(OH)D and risk of mortality may be modified by the intake level of magnesium. Future studies, including cohort studies and clinical trials, are necessary to confirm the findings.
doi:10.1186/1741-7015-11-187
PMCID: PMC3765911  PMID: 23981518
Magnesium intake; Serum 25-hydroxyvitamin D levels; Vitamin D insufficiency; Vitamin D deficiency; Parathyroid hormone; Mortality; Colorectal cancer; Cardiovascular diseases
15.  Postmenopausal hormone therapy is associated with a reduced risk of colorectal cancer lacking CDKN1A expression 
Cancer Research  2012;72(12):3020-3028.
Experimental studies have shown that estrogen- or progesterone-activated signaling leads to growth inhibition effects on colon cancer cells through the upregulation of several cell cycle regulators. However, epidemiologic studies evaluating hormone therapy (HT) use and colorectal cancer risk by the status of cell cycle regulators are lacking. In this study, we used data from the prospective Nurses’ Health Study to evaluate whether the association between HT use and colorectal cancer risk differs by the molecular pathological status of microsatellite instability (MSI) and expression of cell cycle-related tumor biomarkers, including CDKN1A (p21, CIP1), CDKN1B (p27, KIP1), and TP53 (p53) by immunohistochemistry. Duplication Cox regression analysis was used to determine an association between HT use, cancer risk, and specific tumor biomarkers in 581 incident colon and rectal cancer cases that occurred during 26 years of follow-up among 105520 postmenopausal women. We found a difference between HT use and colorectal cancer risk according to CDKN1A expression (p-value for heterogeneity=0.01). Current HT use was associated with a reduced risk for CDKN1A-nonexpressed (multivariate relative risk (RR)=0.61, 95% confidence interval (CI), 0.46–0.82), but not for CDKN1A-expressed (RR=1.32, 95% CI, 0.76–2.31) tumors. The lower risk for CDKN1A-nonexpressed, but not for CDKN1A-expressed cancers was also present among current users of estrogen-alone therapy. We found no significant difference in the relations between HT use and cancer risk according to MSI, CDKN1B, or TP53 status. Together, our molecular epidemiology findings suggest a preventive effect of HT against colorectal carcinogenesis which depends, in part, on loss of cyclin-dependent kinase inhibitor CDKN1A.
doi:10.1158/0008-5472.CAN-11-2619
PMCID: PMC3377852  PMID: 22511578
16.  Combination of HDAC and topoisomerase inhibitors in small cell lung cancer 
Cancer Biology & Therapy  2012;13(8):614-622.
Histone deacetylase (HDAC) inhibitors, including MGCD0103 and vorinostat, have led to tumor growth inhibition and apoptosis in vivo. However, with limited single-agent activity demonstrated in solid tumor trials, we examined the potential for enhanced effects in combination with topoisomerase I and II inhibitors, a staple for treatment in refractory small cell lung cancer (SCLC). SCLC cell lines were exposed to increasing concentrations of single-agent HDAC inhibitors and topoisomerase inhibitors, in various combinations, to assess for cell viability, additivity or synergy, and apoptosis. We found that MGCD0103 and vorinostat decreased cell viability by at least 60% and 80%, respectively. In the majority of cell lines, the strongest synergism was seen when vorinostat was followed by either etoposide or topotecan; concurrent therapy led to antagonism in most cell lines. Synergistic effects were seen when MGCD0103 was given concurrently or sequentially with both amrubicin and epirubicin. Enhanced additive effects leading to caspase activation were noted for the combination of MGCD0103 or vorinostat with a topoisomerase inhibitor vs. either agent alone. Thus, the combination of HDAC inhibitors and topoisomerase inhibitors showed enhanced cytotoxic effects in SCLC cell lines. Further evaluation in a clinical setting may be warranted.
doi:10.4161/cbt.19848
PMCID: PMC3408970  PMID: 22441819
combination therapy; HDAC; SCLC; survival; topoisomerase inhibitors
17.  The VITamin D and OmegA-3 TriaL (VITAL): Rationale and Design of a Large Randomized Controlled Trial of Vitamin D and Marine Omega-3 Fatty Acid Supplements for the Primary Prevention of Cancer and Cardiovascular Disease 
Contemporary Clinical Trials  2011;33(1):159-171.
Data from laboratory studies, observational research, and/or secondary prevention trials suggest that vitamin D and marine omega-3 fatty acids may reduce risk for cancer or cardiovascular disease (CVD), but primary prevention trials with adequate dosing in general populations (i.e., unselected for disease risk) are lacking. The ongoing VITamin D and OmegA-3 TriaL (VITAL) is a large randomized, double-blind, placebo-controlled, 2×2 factorial trial of vitamin D (in the form of vitamin D3 [cholecalciferol], 2000 IU/day) and marine omega-3 fatty acid (Omacor® fish oil, eicosapentaenoic acid [EPA] + docosahexaenoic acid [DHA], 1 g/day) supplements in the primary prevention of cancer and CVD among a multi-ethnic population of 20,000 U.S. men aged ≥50 and women aged ≥55. The mean treatment period will be 5 years. Baseline blood samples will be collected in at least 16,000 participants, with follow-up blood collection in about 6000 participants. Yearly follow-up questionnaires will assess treatment compliance (plasma biomarker measures will also assess compliance in a random sample of participants), use of non-study drugs or supplements, occurence of endpoints, and cancer and vascular risk factors. Self-reported endpoints will be confirmed by medical record review by physicians blinded to treatment assignment, and deaths will be ascertained through national registries and other sources. Ancillary studies will investigate whether these agents affect risk for diabetes and glucose intolerance; hypertension; cognitive decline; depression; osteoporosis and fracture; physical disability and falls; asthma and other respiratory diseases; infections; rheumatoid arthritis, systemic lupus erythematosus, thyroid diseases, and other autoimmune disorders.
doi:10.1016/j.cct.2011.09.009
PMCID: PMC3253961  PMID: 21986389
Cancer; cardiovascular disease; cholecalciferol; primary prevention; omega-3 fatty acids; vitamin D; randomized controlled trial
18.  Sunlight exposure, vitamin D, and risk of non-Hodgkin lymphoma in the Nurses’ Health Study 
Cancer causes & control : CCC  2011;22(12):1731-1741.
Purpose
Case-control studies suggest increased sun exposure reduces non-Hodgkin lymphoma (NHL) risk. Evidence from prospective cohort studies, however, is limited and inconsistent. We evaluated the association between ambient ultraviolet radiation (UV) exposure and NHL in a nationwide cohort of women, the Nurses’ Health Study (NHS).
Methods
Between 1976 and 2006, we identified 1064 incident NHL cases among 115,482 women in the prospective NHS. Exposures assessed included average annual UV-B flux based on residence at various times during life, vitamin D intake, and predicted plasma 25-hydroxyvitamin D levels. We estimated incidence rate ratios (RRs) and 95% confidence intervals (CIs) for risk of all NHL and histologic subtypes using Cox proportional hazards models.
Results
NHL risk was increased for women residing in areas of high ambient UV radiation (UV-B flux >113 R-B count × 10−4) compared to those with lower exposure (<113), with positive linear trends at all time points. The multivariable-adjusted RR for high UV area at age 15 was 1.21 (95% CI: 1.00, 1.47; p-trend <0.01). There was no evidence of statistical heterogeneity by subtype, although power was limited for subtype analyses. We observed no association between vitamin D measures and risk of NHL overall or by subtype.
Conclusions
Our findings do not support the hypothesis of a protective effect of UV radiation exposure on NHL risk. We found no association between vitamin D and NHL risk.
doi:10.1007/s10552-011-9849-x
PMCID: PMC3240999  PMID: 21987081
non-Hodgkin lymphoma; sunlight; ultraviolet radiation; vitamin D; epidemiology
19.  N-Acetyltransferase 2 Polymorphisms, Tobacco Smoking, and Breast Cancer Risk in the Breast and Prostate Cancer Cohort Consortium 
American Journal of Epidemiology  2011;174(11):1316-1322.
Common polymorphisms in the N-acetyltransferase 2 gene (NAT2) modify the association between cigarette smoking and bladder cancer and have been hypothesized to determine whether active cigarette smoking increases breast cancer risk. The authors sought to replicate the latter hypothesis in a prospective analysis of 6,900 breast cancer cases and 9,903 matched controls drawn from 6 cohorts (1989–2006) in the National Cancer Institute’s Breast and Prostate Cancer Cohort Consortium. Standardized methods were used to genotype the 3 most common polymorphisms that define NAT2 acetylation phenotype (rs1799930, rs1799931, and rs1801280). In unconditional logistic regression analyses, breast cancer risk was higher in women with more than 20 pack-years of active cigarette smoking than in never smokers (odds ratio (OR) = 1.28, 95% confidence interval (CI): 1.17, 1.39), after controlling for established risk factors other than alcohol consumption and physical inactivity. However, associations were similar for the slow (OR = 1.25, 95% CI: 1.11, 1.39) and rapid/intermediate (OR = 1.24, 95% CI: 1.08, 1.42) acetylation phenotypes, with no evidence of interaction (P = 0.87). These results provide some support for the hypothesis that long-term cigarette smoking may be causally associated with breast cancer risk but underscore the need for caution when interpreting sparse data on gene-environment interactions.
doi:10.1093/aje/kwr257
PMCID: PMC3390163  PMID: 22074863
arylamine N-acetyltransferase; breast neoplasms; NAT2 protein, human; polymorphism, single nucleotide; smoking
20.  Interactions Between Genetic Variants and Breast Cancer Risk Factors in the Breast and Prostate Cancer Cohort Consortium 
Background
Recently, several genome-wide association studies have identified various genetic susceptibility loci for breast cancer. Relatively little is known about the possible interactions between these loci and the established risk factors for breast cancer.
Methods
To assess interactions between single-nucleotide polymorphisms (SNPs) and established risk factors, we prospectively collected DNA samples and questionnaire data from 8576 breast cancer case subjects and 11 892 control subjects nested within the National Cancer Institute’s Breast and Prostate Cancer Cohort Consortium (BPC3). We genotyped 17 germline SNPs (FGFR2-rs2981582, FGFR2-rs3750817, TNRC9-rs3803662, 2q35-rs13387042, MAP3K1-rs889312, 8q24-rs13281615, CASP8-rs1045485, LSP1-rs3817198, COL1A1-rs2075555, COX11-rs6504950, RNF146-rs2180341, 6q25-rs2046210, SLC4A7-rs4973768, NOTCH2-rs11249433, 5p12-rs4415084, 5p12-rs10941679, RAD51L1-rs999737), and odds ratios were estimated by logistic regression to confirm previously reported associations with breast cancer risk. We performed likelihood ratio test to assess interactions between 17 SNPs and nine established risk factors (age at menarche, parity, age at menopause, use of hormone replacement therapy, family history, height, body mass index, smoking status, and alcohol consumption), and a correction for multiple testing of 153 tests (adjusted P value threshold = .05/153 = 3 × 10−4) was done. Case–case comparisons were performed for possible differential associations of polymorphisms by subgroups of tumor stage, estrogen and progesterone receptor status, and age at diagnosis. All statistical tests were two-sided.
Results
We confirmed the association of 14 SNPs with breast cancer risk (Ptrend = 2.57 × 10−3 –3.96 × 10−19). Three SNPs (LSP1-rs3817198, COL1A1-rs2075555, and RNF146-rs2180341) did not show association with breast cancer risk. After accounting for multiple testing, no statistically significant interactions were detected between the 17 SNPs and the nine risk factors. We also confirmed that SNPs in FGFR2 and TNRC9 were associated with greater risk of estrogen receptor–positive than estrogen receptor–negative breast cancer (Pheterogeneity = .0016 for FGFR2-rs2981582 and Pheterogeneity = .0053 for TNRC9-rs3803662). SNP 5p12-rs10941679 was statistically significantly associated with greater risk of progesterone receptor–positive than progesterone receptor–negative breast cancer (Pheterogeneity = .0028).
Conclusion
This study does not support the hypothesis that known common breast cancer susceptibility loci strongly modify the associations between established risk factors and breast cancer.
doi:10.1093/jnci/djr265
PMCID: PMC3156803  PMID: 21791674
21.  The Aromatase Gene (CYP19A1) Variants and Circulating Hepatocyte Growth Factor in Postmenopausal Women 
PLoS ONE  2012;7(7):e42079.
Background
Estrogen and androgen have been linked to the regulation of circulating hepatocyte growth factor (HGF), an adipose tissue-derived cytokine. It is possible that the CYP19A1 gene which alters sex hormones production may influence HGF levels. We examined the association between the CYP19A1 gene variants and plasma HGF concentrations.
Design
We evaluated 45 common and putative functional variants of CYP19A1 and circulating levels of HGF among 260 postmenopausal women who later developed colorectal cancer from the Women's Health Initiative Observational Cohort. As the distribution of HGF levels was highly skewed, we transformed HGF concentrations for all women into a log-, ranked-, or normal score-scale value. Multiple linear regression with adjustment for age was used to evaluate the associations.
Results
We observed an association between the rs7172156, rs1008805, rs6493494, rs749292, and rs11636639 variants and HGF levels in ranked and normal score scales (corrected p values ≤0.02), although the association of these 5 SNPs with log-scale HGF was not significant (corrected p values ≥0.16). The associations remained unchanged after additional adjustment for hormone therapy use and estradiol levels. These 5 SNPs, which were in linkage disequilibrium (pairwise D′≥97%, r2≥56%), constituted a block with 2 common haplotypes accounting for 82% frequency. The most common haplotype, TCCCA, was associated with lower ranked- or normal score-transformed HGF levels (corrected p values ≤0.001), whereas the second most common haplotype, CTTCA, was associated with higher ranked- or normal score-transformed HGF levels (corrected p values ≤0.02).
Conclusion
Our findings of a potential association between the CYP19A1 variants and circulating HGF levels warrant confirmation in studies with larger sample size.
doi:10.1371/journal.pone.0042079
PMCID: PMC3405042  PMID: 22848710
22.  Stable silencing of β-lactoglobulin (BLG) gene by lentivirus-mediated RNAi in goat fetal fibroblasts 
Genetics and Molecular Biology  2012;35(3):680-685.
β-lactoglobulin (BLG), a dominant allergen in goat milk, is difficult to remove by traditional biochemical methods. Its elimination from goat milk by genetic modification therefore poses a major challenge for modern goat breeders. A shRNA targeting BLG mRNA with high interference efficiency was identified, with which lentiviral vectors were used for mediating stable shRNA interference in goat-fetal fibroblast cells. Apart from high efficiency in the knockdown of BLG expression in these cells, lentivector-mediated RNAi manifested stable integration into the goat genome itself. Consequently, an in vitro model for goat BLG-content control was compiled, and a goat-cell line for accompanying transgenetic goat production created.
doi:10.1590/S1415-47572012005000042
PMCID: PMC3459420  PMID: 23055809
RNAi; shRNA; lentivirus; BLG; goat
23.  EPLIN Downregulation Promotes Epithelial-Mesenchymal Transition in Prostate Cancer Cells and Correlates With Clinical Lymph Node Metastasis 
Oncogene  2011;30(50):4941-4952.
Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling, and activation of β-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosing and treating metastasis at early stages.
doi:10.1038/onc.2011.199
PMCID: PMC3165108  PMID: 21625216
EPLIN; epithelial-mesenchymal transition; prostate cancer; lymph node metastasis; cytoskeleton
24.  Prediction and Identification of Potential Immunodominant Epitopes in Glycoproteins B, C, E, G, and I of Herpes Simplex Virus Type 2 
Twenty B candidate epitopes of glycoproteins B (gB2), C (gC2), E (gE2), G (gG2), and I (gI2) of herpes simplex virus type 2 (HSV-2) were predicted using DNAstar, Biosun, and Antheprot methods combined with the polynomial method. Subsequently, the biological functions of the peptides were tested via experiments in vitro. Among the 20 epitope peptides, 17 could react with the antisera to the corresponding parent proteins in the EIA tests. In particular, five peptides, namely, gB2466–473 (EQDRKPRN), gC2216–223 (GRTDRPSA), gE2483–491 (DPPERPDSP), gG2572–579 (EPPDDDDS), and gI2286-295 (CRRRYRRPRG) had strong reaction with the antisera. All conjugates of the five peptides with the carrier protein BSA could stimulate mice into producing antibodies. The antisera to these peptides reacted strongly with the corresponding parent glycoproteins during the Western Blot tests, and the peptides reacted strongly with the antibodies against the parent glycoproteins during the EIA tests. The antisera against the five peptides could neutralize HSV-2 infection in vitro, which has not been reported until now. These results suggest that the immunodominant epitopes screened using software algorithms may be used for virus diagnosis and vaccine design against HSV-2.
doi:10.1155/2012/205313
PMCID: PMC3357521  PMID: 22649465
25.  A Phase I Clinical-Pharmacodynamic Study of the Farnesyltransferase Inhibitor Tipifarnib in Combination with the Proteasome Inhibitor Bortezomib in Advanced Acute Leukemias 
Purpose
To determine the safety, target inhibition, and signals of clinical activity of tipifarnib in combination with bortezomib in patients with advanced acute leukemias.
Experimental Design
In a 3+3 design, patients received escalating doses of tipifarnib (days 1–14) and bortezomib (days 1, 4, 8, 11) every 3 weeks until maximum tolerated dose was reached. Peripheral blood mononuclear cells (PBMCs) were collected at days 1, 8, and 22 for measurement of chymotrypsin-like and farnesyltransferase activity. Purified bone marrow leukemic blasts were collected at baseline and at day 8 for measurement of NF-kB activity.
Results
The combination was well-tolerated, and maximum tolerated dose was not reached. Dose-limiting toxicities included diarrhea, fatigue, and sensorimotor neuropathy. Chymotrypsin-like and farnesyltransferase activity within PBMCs were decreased in a majority of patients at day 8. NF-kB activity within leukemic blasts was decreased in a majority of patients at day 8. Complete response with incomplete count recovery was observed in 2 patients, and an additional 5 patients had stable disease.
Conclusions
Tipifarnib and bortezomib combination in patients with advanced leukemias was well-tolerated, demonstrated relevant target inhibition, and was associated with signals of clinical activity in patients with advanced and refractory acute leukemias. Future studies of this combination may be warranted in more selected groups of patients in whom these molecular targets are of particular importance.
doi:10.1158/1078-0432.CCR-10-1878
PMCID: PMC3049960  PMID: 21233404
acute leukemia; NF-kappa B; farnesyltransferase inhibitor; proteasome inhibitor; phase 1

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