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1.  Vegetarian diets and cardiovascular risk factors in black members of the Adventist Health Study-2 
Public health nutrition  2014;18(3):537-545.
Objective
To compare cardiovascular risk factors between vegetarians and non-vegetarians in black individuals living in the USA.
Design
A cross-sectional analysis of a sub-set of 592 black women and men enrolled in the Adventist Health Study-2 (AHS-2) cohort of Seventh-day Adventists.
Setting
Members of the AHS-2 cohort, who lived in all states of the USA and provinces of Canada.
Subjects
Black/African-American members of two sub-studies of AHS-2 where blood and physiological measurements were obtained.
Results
Of these women and men, 25% were either vegan or lacto-ovo-vegetarians (labelled ‘vegetarian/vegans’), 13 % were pesco-vegetarian and 62% were non-vegetarian. Compared with non-vegetarians, the vegetarian/vegans had odds ratios for hypertension, diabetes, high blood total cholesterol and high blood LDL-cholesterol of 0·56 (95% CI 0·36, 0·87), 0·48 (95% CI 0·24, 0·98), 0·42 (95% CI 0·27, 0·65) and 0·54 (95% CI 0·33, 0·89), respectively, when adjusted for age, gender, education, physical activity and sub-study. Corresponding odds ratios for obesity in vegetarian/vegans and pesco-vegetarians, compared with non-vegetarians, were 0·43 (95% CI 0·28, 0·67) and 0·47 (95% CI 0·27, 0·81), respectively; and for abdominal obesity 0·54 (95% CI 0·36, 0·82) and 0·50 (95% CI 0·29, 0·84), respectively. Results for pesco-vegetarians did not differ significantly from those of non-vegetarians for other variables. Further adjustment for BMI suggested that BMI acts as an intermediary variable between diet and both hypertension and diabetes.
Conclusions
As with non-blacks, these results suggest that there are sizeable advantages to a vegetarian diet in black individuals also, although a cross-sectional analysis cannot conclusively establish cause.
doi:10.1017/S1368980014000263
PMCID: PMC4167463  PMID: 24636393
Vegetarian diet; Cardiovascular risks; Blacks; Adventists
2.  Copy Number Variation Analysis by Ligation-Dependent PCR Based on Magnetic Nanoparticles and Chemiluminescence 
Theranostics  2015;5(1):71-85.
A novel system for copy number variation (CNV) analysis was developed in the present study using a combination of magnetic separation and chemiluminescence (CL) detection technique. The amino-modified probes were firstly immobilized onto carboxylated magnetic nanoparticles (MNPs) and then hybridized with biotin-dUTP products, followed by amplification with ligation-dependent polymerase chain reaction (PCR). After streptavidin-modified alkaline phosphatase (STV-AP) bonding and magnetic separation, the CL signals were then detected. Results showed that the quantification of PCR products could be reflected by CL signal values. Under optimum conditions, the CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with logarithm of the target concentration. To validate the methodology, copy numbers of six genes from the human genome were detected. To compare the detection accuracy, multiplex ligation-dependent probe amplification (MLPA) and MNPs-CL detection were performed. Overall, there were two discrepancies by MLPA analysis, while only one by MNPs-CL detection. This research demonstrated that the novel MNPs-CL system is a useful analytical tool which shows simple, sensitive, and specific characters which are suitable for CNV analysis. Moreover, this system should be improved further and its application in the genome variation detection of various diseases is currently under further investigation.
doi:10.7150/thno.10117
PMCID: PMC4265749  PMID: 25553099
Copy number variation; Ligation-dependent PCR; Magnetic nanoparticles; Chemiluminescence.
3.  Quantitative flux analysis reveals folate-dependent NADPH production 
Nature  2014;510(7504):298-302.
ATP is the dominant energy source in animals for mechanical and electrical work (e.g., muscle contraction, neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defense and reductive biosynthesis1. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway (oxPPP), with malic enzyme sometimes also important. While the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analyzed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labeled substrates into NADPH, and combine this approach with carbon labeling and mathematical modeling to measure cytosolic NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxPPP. Surprisingly a nearly comparable contribution comes from serine-driven one-carbon metabolism, where oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. Since folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and GSH/GSSG ratios and increased cell sensitivity to oxidative stress. Thus, while the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.
doi:10.1038/nature13236
PMCID: PMC4104482  PMID: 24805240
4.  Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate 
Cancer & Metabolism  2014;2:23.
Background
Cell growth requires fatty acids for membrane synthesis. Fatty acids are assembled from 2-carbon units in the form of acetyl-CoA (AcCoA). In nutrient and oxygen replete conditions, acetyl-CoA is predominantly derived from glucose. In hypoxia, however, flux from glucose to acetyl-CoA decreases, and the fractional contribution of glutamine to acetyl-CoA increases. The significance of other acetyl-CoA sources, however, has not been rigorously evaluated. Here we investigate quantitatively, using 13C-tracers and mass spectrometry, the sources of acetyl-CoA in hypoxia.
Results
In normoxic conditions, cultured cells produced more than 90% of acetyl-CoA from glucose and glutamine-derived carbon. In hypoxic cells, this contribution dropped, ranging across cell lines from 50% to 80%. Thus, under hypoxia, one or more additional substrates significantly contribute to acetyl-CoA production. 13C-tracer experiments revealed that neither amino acids nor fatty acids are the primary source of this acetyl-CoA. Instead, the main additional source is acetate. A large contribution from acetate occurs despite it being present in the medium at a low concentration (50–500 μM).
Conclusions
Acetate is an important source of acetyl-CoA in hypoxia. Inhibition of acetate metabolism may impair tumor growth.
Electronic supplementary material
The online version of this article (doi:10.1186/2049-3002-2-23) contains supplementary material, which is available to authorized users.
doi:10.1186/2049-3002-2-23
PMCID: PMC4322440  PMID: 25671109
Acetate; Acetyl-CoA; Cancer metabolism; Fatty acids; Hypoxia; Lipogenesis; Mass spectrometry; Palmitate; 13C-tracing
5.  Reciprocal Changes in Vanilloid (TRPV1) and Endocannabinoid (CB1) Receptors Contribute to Visceral Hyperalgesia in the Water Avoidance Stressed Rat 
Gut  2008;58(2):202-210.
Background
Increasing evidence suggests that chronic stress plays an important role in the pathophysiology of several functional gastrointestinal disorders. We investigated whether CB1 and TRPV1 receptors are involved in stress induced visceral hyperalgesia.
Methods
Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Immunofluorescence and Western blot analysis were used to assess the expression of CB1 and TRPV1 receptors in DRG neurons.
Results
WA stressed rats demonstrated a significant increase in the serum corticosterone levels and fecal pellet output compared to controls supporting stimulation of the HPA axis. The VMR increased significantly at pressures of 40 and 60 mmHg in WA stress rats compared with controls, respectively, and was associated with hyperalgesia. The endogenous CB1 agonist anandamide was increased significantly in DRGs from stressed rats. Immunofluorescence and Western blot analysis showed a significant decrease in CB1 and reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide in vitro. In contrast, treatment of control DRGs in vitro with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats in situ with WIN or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the up-regulation of TRPV1.
Conclusions
These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia.
doi:10.1136/gut.2008.157594
PMCID: PMC4236191  PMID: 18936104
TRPV1; CB1; visceral hyperalgesia; stress; dorsal root ganglion
6.  Patterns of food consumption among vegetarians and non-vegetarians 
The British journal of nutrition  2014;112(10):1644-1653.
Vegetarian dietary patterns have been reported to be associated with a number of favourable health outcomes in epidemiological studies, including the Adventist Health Study 2 (AHS-2). Such dietary patterns may vary and need further characterisation regarding foods consumed. The aims of the present study were to characterise and compare the food consumption patterns of several vegetarian and non-vegetarian diets. Dietary intake was measured using an FFQ among more than 89 000 members of the AHS-2 cohort. Vegetarian dietary patterns were defined a priori, based on the absence of certain animal foods in the diet. Foods were categorised into fifty-eight minor food groups comprising seventeen major food groups. The adjusted mean consumption of each food group for the vegetarian dietary patterns was compared with that for the non-vegetarian dietary pattern. Mean consumption was found to differ significantly across the dietary patterns for all food groups. Increased consumption of many plant foods including fruits, vegetables, avocados, non-fried potatoes, whole grains, legumes, soya foods, nuts and seeds was observed among vegetarians. Conversely, reduced consumption of meats, dairy products, eggs, refined grains, added fats, sweets, snack foods and non-water beverages was observed among vegetarians. Thus, although vegetarian dietary patterns in the AHS-2 have been defined based on the absence of animal foods in the diet, they differ greatly with respect to the consumption of many other food groups. These differences in food consumption patterns may be important in helping to explain the association of vegetarian diets with several important health outcomes.
doi:10.1017/S000711451400261X
PMCID: PMC4232985  PMID: 25247790
Dietary patterns; Vegetarians; Foods
7.  Vegetarian Dietary Patterns and Mortality in Adventist Health Study 2 
JAMA internal medicine  2013;173(13):1230-1238.
Importance
Some evidence suggests vegetarian dietary patterns may be associated with reduced mortality, but the relationship is not well established.
Objective
To evaluate the association between vegetarian dietary patterns and mortality.
Design
Prospective cohort study; mortality analysis by Cox proportional hazards regression, controlling for important demographic and lifestyle confounders.
Setting
Adventist Health Study 2 (AHS-2), a large North American cohort.
Participants
A total of 96 469 Seventh-day Adventist men and women recruited between 2002 and 2007, from which an analytic sample of 73 308 participants remained after exclusions.
Exposures
Diet was assessed at baseline by a quantitative food frequency questionnaire and categorized into 5 dietary patterns: nonvegetarian, semi-vegetarian, pesco-vegetarian, lacto-ovo–vegetarian, and vegan.
Main Outcome and Measure
The relationship between vegetarian dietary patterns and all-cause and cause-specific mortality; deaths through 2009 were identified from the National Death Index.
Results
There were 2570 deaths among 73 308 participants during a mean follow-up time of 5.79 years. The mortality rate was 6.05 (95% CI, 5.82–6.29) deaths per 1000 person-years. The adjusted hazard ratio (HR) for all-cause mortality in all vegetarians combined vs non-vegetarians was 0.88 (95% CI, 0.80–0.97). The adjusted HR for all-cause mortality in vegans was 0.85 (95% CI, 0.73–1.01); in lacto-ovo–vegetarians, 0.91 (95% CI, 0.82–1.00); in pesco-vegetarians, 0.81 (95% CI, 0.69–0.94); and in semi-vegetarians, 0.92 (95% CI, 0.75–1.13) compared with nonvegetarians. Significant associations with vegetarian diets were detected for cardiovascular mortality, noncardiovascular noncancer mortality, renal mortality, and endocrine mortality. Associations in men were larger and more often significant than were those in women.
Conclusions and Relevance
Vegetarian diets are associated with lower all-cause mortality and with some reductions in cause-specific mortality. Results appeared to be more robust in males. These favorable associations should be considered carefully by those offering dietary guidance.
doi:10.1001/jamainternmed.2013.6473
PMCID: PMC4191896  PMID: 23836264
8.  Solution Structure and Dynamics of Human Hemoglobin in the Carbonmonoxy Form 
Biochemistry  2013;52(34):5809-5820.
The solution structure of HbCO A was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous NMR structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution with low-salt resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A. Amide-water proton exchange experiments demonstrated that αThr38 located in the α1β2 interface and several loop regions in both α- and β-chains were dynamic on the sub-second timescale. Side-chain methyl dynamics revealed that methyl groups in the α1β2 interface were dynamic, but those in the α1β1 interface were quite rigid on the ns-ps and ms-µs timescales. All the data strongly suggest a dynamic α1β2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamics changes in the α1β2 interface and the regions around the hemes, but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the ms timescale, very likely arising from the relative motion of the α1β1 dimer with respect to the α2β2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions.
doi:10.1021/bi4005683
PMCID: PMC4013309  PMID: 23901897
9.  Estimating the ROC Curve in Studies that Match Controls to Cases on Covariates 
Academic radiology  2013;20(7):863-873.
Rationale and Objectives
Studies evaluating a new diagnostic imaging test may select control subjects without disease who are similar to case subjects with disease in regards to factors potentially related to the imaging result. Selecting one or more controls that are matched to each case on factors such as age, co-morbidities or study site improves study validity by eliminating potential biases due to differential characteristics of readings for cases versus controls. However it is not widely appreciated that valid analysis requires that the receiver operating characteristic (ROC) curve be adjusted for covariates. We propose a new computationally simple method for estimating the covariate adjusted ROC curve that is appropriate in matched case-control studies.
Materials and Methods
We provide theoretical arguments for the validity of the estimator and demonstrate its application to data. We compare the statistical properties of the estimator with those of a previously proposed estimator of the covariate adjusted ROC curve. We demonstrate an application of the estimator to data derived from a study of emergency medical services (EMS) encounters where the goal is to diagnose critical illness in non-trauma, non-cardiac arrest patients. A novel bootstrap method is proposed for calculating confidence intervals.
Results
The new estimator is computationally very simple, yet we show it yields values that approximate the existing, more complicated estimator in simulated data sets. We found that the new estimator has excellent statistical properties, with bias and efficiency comparable with the existing method.
Conclusion
In matched case-control studies the ROC curve should be adjusted for matching covariates and can be estimated with the new computationally simple approach.
doi:10.1016/j.acra.2013.03.004
PMCID: PMC3679266  PMID: 23601953
risk prediction; logistic regression; diagnostic test; classification; case-control study
10.  Tibial lengthening for unilateral Crowe type-IV developmental dysplasia of the hip 
Indian Journal of Orthopaedics  2014;48(4):415-420.
Background:
Developmental dysplasia of the hip (DDH) is associated with chronic pain and limping which especially has a negative impact on the patients’ daily activities, body image, and self-esteem. Although total hip arthroplasty remains the first choice for treatment of DDH in adults, minimally invasive alternative approaches are being increasingly favored both by the surgeon and the patients with severe DDH. This study aimed to evaluate the outcome of these patients treated with a mono-lateral external fixator-based tibial lengthening procedure.
Materials and Methods:
During the period of month between June 1999 and January 2006, 13 (mean ages 20.8 years) adult patients with unilateral Crowe type-IV DDH were treated by tibial lengthening using a mono-lateral external fixator over an intramedullary nail. Bone healing, infection, gait correction and improvement in body image were assessed during postoperative followup. Patients’ overall health status at the end of followup was assessed using the short form-36 (SF-36) health survey.
Results:
Patients were followed up for an average of 7.3 years. Successful bone healing was observed in all 13 patients and no further surgeries were indicated. A mean external fixation index of 12.4 days/cm was achieved. Bone formation fell in good to excellent categories with a mean consolidation index of 50.1 days/cm. Pin-tract infections were observed in two patients. The degree of limping was reduced from severe or moderate preoperatively to mild postoperatively. Neither equinus deformity nor painful degenerative osteoarthritis and hip dysfunction were observed in any of the patients studied. The SF-36 questionnaire survey showed that all patients were satisfied with their outcomes.
Conclusions:
Tibial lengthening may effectively correct gait and satisfactorily improve body image in young patients with unilateral Crowe type-IV DDH. Mono-lateral external fixator allows for accelerated postoperative rehabilitation and optimal preservation of ankle movements. Lengthening along with intramedullary nails may significantly reduce the external fixation time and the risk of fixator-related complications.
doi:10.4103/0019-5413.136302
PMCID: PMC4137522  PMID: 25143648
Developmental dysplasia of the hip; intramedullary nail; tibial lengthening; Hip dysplasia; intramedullary nailing; tibia
11.  Quantitation of Cellular Metabolic Fluxes of Methionine 
Analytical chemistry  2014;86(3):1583-1591.
Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosyn-thesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with 13C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography−mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools.
doi:10.1021/ac4032093
PMCID: PMC4060246  PMID: 24397525
12.  Dose-Dependent Effect of Estrogen Suppresses the Osteo-Adipogenic Transdifferentiation of Osteoblasts via Canonical Wnt Signaling Pathway 
PLoS ONE  2014;9(6):e99137.
Fat infiltration within marrow cavity is one of multitudinous features of estrogen deficiency, which leads to a decline in bone formation functionality. The origin of this fat is unclear, but one possibility is that it is derived from osteoblasts, which transdifferentiate into adipocytes that produce bone marrow fat. We examined the dose-dependent effect of 17β-estradiol on the ability of MC3T3-E1 cells and murine bone marrow-derived mesenchymal stem cell (BMMSC)-derived osteoblasts to undergo osteo-adipogenic transdifferentiation. We found that 17β-estradiol significantly increased alkaline phosphatase activity (P<0.05); calcium deposition; and Alp, Col1a1, Runx2, and Ocn expression levels dose-dependently. By contrast, 17β-estradiol significantly decreased the number and size of lipid droplets, and Fabp4 and PPARγ expression levels during osteo-adipogenic transdifferentiation (P<0.05). Moreover, the expression levels of brown adipocyte markers (Myf5, Elovl3, and Cidea) and undifferentiated adipocyte markers (Dlk1, Gata2, and Wnt10b) were also affected by 17β-estradiol during osteo-adipogenic transdifferentiation. Western blotting and immunostaining further showed that canonical Wnt signaling can be activated by estrogen to exert its inhibitory effect of osteo-adipogenesis. This is the first study to demonstrate the dose-dependent effect of 17β-estradiol on the osteo-adipogenic transdifferentiation of MC3T3-E1 cells and BMMSCs likely via canonical Wnt signaling. In summary, our results indicate that osteo-adipogenic transdifferentiation modulated by canonical Wnt signaling pathway in bone metabolism may be a new explanation for the gradually increased bone marrow fat in estrogen-inefficient condition.
doi:10.1371/journal.pone.0099137
PMCID: PMC4053448  PMID: 24918446
13.  Quantification of NADPH balance during adipogenesis 
Cancer & Metabolism  2014;2(Suppl 1):P40.
doi:10.1186/2049-3002-2-S1-P40
PMCID: PMC4072990
16.  A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18 
Nucleic Acids Research  2014;42(11):7305-7318.
Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.
doi:10.1093/nar/gku350
PMCID: PMC4066777  PMID: 24782531
17.  Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway 
Molecular and Cellular Biochemistry  2014;392(1-2):85-93.
Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5′-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.
Electronic supplementary material
The online version of this article (doi:10.1007/s11010-014-2021-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s11010-014-2021-7
PMCID: PMC4053611  PMID: 24390089
Estrogen; Postmenopausal osteoporosis; HBMSCs; Notch signaling pathway; Cell proliferation; Cell differentiation
18.  Soy isoflavone intake and the likelihood of ever becoming a mother: the Adventist Health Study-2 
Objectives
As little is known about the possible relationship between the intake of phytoestrogens and female fertility, we investigated the relationship between soy isoflavone intake and the risk of nulliparity and nulligravidity.
Methods
A cross-sectional study of 11,688 North American Adventist women aged 30–50 years old with data regarding childbearing. These women were, as a group, characterized by a high proportion (54%) of vegetarians and a healthy lifestyle with a very low prevalence of smoking and alcohol use.
Results
The mean isoflavone intake (17.9 mg per day) was very high compared to other Western populations. Only 6% of the women indicated no intake of isoflavones. We found, after adjustment for age, marital status, and educational level, an inverse relationship (P=0.05) between isoflavone intake and the likelihood of ever having become a mother. In women with high (≥40 mg/day) isoflavone intake (12% of this group of women), the adjusted lifetime probability of giving birth to a live child was reduced by approximately 3% (95% CI: 0, 7) compared to women with low (<10 mg/day) intake. No relationships were found between the isoflavone intake and parity or age at first delivery in parous women. A similar inverse relationship (P=0.03) was found between the isoflavone intake and the risk of nulligravidity with a 13% (95% CI: 2, 26) higher risk of never have been pregnant in women with high (≥40 mg/day) isoflavone intake. These relationships were found mainly in women who reported problems becoming pregnant.
Conclusion
The findings suggest that a high dietary isoflavone intake may have significant impact on fertility.
doi:10.2147/IJWH.S57137
PMCID: PMC3982974  PMID: 24741329
soybeans; phytoestrogens; isoflavones; fertility; Seventh-Day Adventist
19.  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.
doi:10.1371/journal.pone.0091388
PMCID: PMC3956664  PMID: 24637927
20.  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.
doi:10.1371/journal.pone.0090343
PMCID: PMC3948675  PMID: 24614680
21.  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.
doi:10.1371/journal.pone.0090220
PMCID: PMC3942417  PMID: 24595049
22.  VEGETARIAN DIETS AND THE INCIDENCE OF CANCER IN A LOW-RISK POPULATION 
Background
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.
Methods
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.
Results
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).
Conclusion
Vegetarian diets seem to confer protection against cancer.
Impact
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.
doi:10.1158/1055-9965.EPI-12-1060
PMCID: PMC3565018  PMID: 23169929
cancer; vegetarian dietary patterns; diet; vegan; lacto-ovo-vegetarians
23.  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
24.  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.
doi:10.3233/JHD-2012-129000
PMCID: PMC3864911  PMID: 24353748
Huntington’s disease; medium spiny neuron; striatum; YAC128; cdk5; p25; quinolinic acid; NMDA; excitotoxic; neurodegeneration
25.  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.
doi:10.1038/msb.2013.65
PMCID: PMC3882799  PMID: 24301801
cancer bioenergetics; isotope tracing; metabolic flux analysis

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