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1.  Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue 
PLoS ONE  2015;10(7):e0127353.
Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.
doi:10.1371/journal.pone.0127353
PMCID: PMC4519244  PMID: 26222067
2.  Thermally switchable polymers achieve controlled Escherichia coli detachment 
Advanced healthcare materials  2014;3(7):1020-1025.
doi:10.1002/adhm.201300518
PMCID: PMC4107026  PMID: 24497458
Switchable; thermally responsive; bacteria; ToF-SIMS; Escherichia coli
3.  Genome-Wide Evaluation of the Interplay between Caenorhabditis elegans and Yersinia pseudotuberculosis during In Vivo Biofilm Formation 
Infection and Immunity  2014;83(1):17-27.
The formation of an incapacitating biofilm on Caenorhabditis elegans by Yersinia pseudotuberculosis represents a tractable model for investigating the genetic basis for host-pathogen interplay during the biofilm-mediated infection of a living surface. Previously we established a role for quorum sensing (QS) and the master motility regulator, FlhDC, in biofilm formation by Y. pseudotuberculosis on C. elegans. To obtain further genome-wide insights, we used transcriptomic analysis to obtain comparative information on C. elegans in the presence and absence of biofilm and on wild-type Y. pseudotuberculosis and Y. pseudotuberculosis QS mutants. Infection of C. elegans with the wild-type Y. pseudotuberculosis resulted in the differential regulation of numerous genes, including a distinct subset of nematode C-lectin (clec) and fatty acid desaturase (fat) genes. Evaluation of the corresponding C. elegans clec-49 and fat-3 deletion mutants showed delayed biofilm formation and abolished biofilm formation, respectively. Transcriptomic analysis of Y. pseudotuberculosis revealed that genes located in both of the histidine utilization (hut) operons were upregulated in both QS and flhDC mutants. In addition, mutation of the regulatory gene hutC resulted in the loss of biofilm, increased expression of flhDC, and enhanced swimming motility. These data are consistent with the existence of a regulatory cascade in which the Hut pathway links QS and flhDC. This work also indicates that biofilm formation by Y. pseudotuberculosis on C. elegans is an interactive process during which the initial attachment/recognition of Yersinia to/by C. elegans is followed by bacterial growth and biofilm formation.
doi:10.1128/IAI.00110-14
PMCID: PMC4288884  PMID: 25312958
4.  Detection of myeloma in skeleton of mice by whole-body optical fluorescence imaging 
Molecular cancer therapeutics  2007;6(6):1701-1708.
Development of new therapies for myeloma has been hindered by the lack of suitable preclinical animal models of the disease in which widespread tumor foci in the skeleton can be detected reliably. Traditional means of detecting skeletal tumor infiltration such as histopathology are cumbersome and labor-intensive and do not allow temporal monitoring of tumor progression or regression in response to therapy. To resolve this problem, we modified the Radl 5TGM1 model of myeloma bone disease such that fluorescent myeloma tumors can be optically imaged in situ. Here, we show that murine myeloma 5TGM1 tumor cells, engineered to express enhanced green fluorescent protein (eGFP; 5TGM1-eGFP cells), can be imaged in a temporal fashion using a fluorescence illuminator and a charge-coupled device camera in skeletons of live C57BL/KaLwRij mice. High-resolution, whole-body images of tumor-bearing mice revealed that myeloma cells homed almost exclusively to the skeleton, with multiple focal tumor foci in the axial skeleton, consistent with myeloma tumor distribution in humans. Finally, the tested antitumor treatment effect of Velcade (bortezomib), a proteasome inhibitor used clinically in myeloma, was readily detected by GFP imaging, suggesting the power of the technique in combination with the Radl 5TGM1-eGFP model for rapid preclinical assessment and sensitive monitoring of novel and potential therapeutics. Whole-body GFP imaging is practical, convenient, inexpensive, and rapid, and these advantages should enable a high throughput when evaluating in vivo efficacy of new potential antimyeloma therapeutics and assessing response to treatment.
doi:10.1158/1535-7163.MCT-07-0121
PMCID: PMC4482358  PMID: 17541032
5.  Evidence of tradeoffs shaping virulence evolution in an emerging wildlife pathogen 
Journal of evolutionary biology  2014;27(6):1271-1278.
In the mid-1990's, the common poultry pathogen Mycoplasma gallisepticum (MG) made a successful species jump to the eastern North American house finch Haemorhous mexicanus (HM). Subsequent strain diversification allows us to directly quantify, in an experimental setting, the transmission dynamics of three sequentially emergent geographic isolates of MG, which differ in the levels of pathogen load they induce. We find significant among-strain variation in rates of transmission as well as recovery. Pathogen strains also differ in their induction of host morbidity, measured as the severity of eye lesions due to infection. Relationships between pathogen traits are also investigated, with transmission and recovery rates being significantly negatively correlated, while transmission and virulence, measured as average eye lesion score over the course of infection, are positively correlated. By quantifying these disease-relevant parameters and their relationships, we provide the first analysis of the tradeoffs that shape the evolution of this important emerging pathogen.
doi:10.1111/jeb.12379
PMCID: PMC4093834  PMID: 24750277
house finch; virulence; transmission; tradeoff; maximum likelihood; cage experiment; eye lesions
6.  Reduced total and cause-specific mortality from walking and running in diabetes 
Objective
This study aimed to assess the relationships of running and walking to mortality in diabetic subjects.
Research design and methods
We studied the mortality surveillance between January 1, 1989 and December 31, 2008, of 2160 participants of the National Walkers' and Runners' Health Studies who reported using diabetic medications at baseline. Hazard ratios (HR) and 95% confidence intervals (95% CI) were obtained from Cox proportional hazard analyses for mortality versus exercise energy expenditure (metabolic equivalents-hours/d or MET-hours/d, 1 MET-hour ~one km run or a 1.5 km brisk walk).
Results
Three hundred and thirty-one diabetic individuals died during a 9.8-yr average follow-up. Merely meeting the current exercise recommendations was not associated with lower all-cause mortality (P = 0.61), whereas exceeding the recommendations was associated with lower all-cause mortality (HR = 0.64, 95% CI = 0.49–0.82, P = 0.0005). Greater MET-hours per day ran or walked was associated with 40% lower risk for all chronic kidney disease-related deaths (HR = 0.60 per MET-hour/d, 95% CI = 0.35–0.91, P = 0.02), 31% lower risk for all sepsis-related deaths (HR = 0.69, 0.47–0.94, P = 0.01), and 31% lower risk for all pneumonia and influenza-related deaths (HR = 0.69, 95% CI = 0.45–0.97, P = 0.03). Running or walking >=1.8 MET-hour/d was associated with 57% reduction in cardiovascular disease (CVD) as an underlying cause of death and 46% lower risk for all CVD-related deaths versus ≤1.07 MET-hours/d. All results remained significant: 1) adjusted for baseline BMI and 2) excluding all deaths within 3 yr of baseline.
Conclusions
These results suggest that 1) exercise is associated with significantly lower all-cause, CVD, chronic kidney disease, sepsis, and pneumonia, and influenza mortality in diabetic patients and 2) higher exercise standards may be warranted for diabetic patients than currently provided to the general population.
doi:10.1249/MSS.0000000000000197
PMCID: PMC4157907  PMID: 24968127
7.  Development and Validation of a Scalable Next-Generation Sequencing System for Assessing Relevant Somatic Variants in Solid Tumors12 
Neoplasia (New York, N.Y.)  2015;17(4):385-399.
Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with < 20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.
doi:10.1016/j.neo.2015.03.004
PMCID: PMC4415141  PMID: 25925381
AOHC, AcroMetrix Oncology Hotspot Control; CNAs, copy number alterations; FFPE, formalin-fixed paraffin-embedded; GoF, gain-of-function; indels, insertions/deletions; LoF, loss-of-function; LU, lung cohort; MCR, minimal common region; MO, molecular cohort; NCCN, National Comprehensive Cancer Network; NGS, next-generation sequencing; OCP, Oncomine Comprehensive Panel; PGM, Personal Genome Machine; PR, prostate cohort; QMRS, Quantitative Multiplex Reference Standard; SCC, small cell carcinoma; TCGA, The Cancer Genome Atlas
8.  Bacterial Attachment to Polymeric Materials Correlates with Molecular Flexibility and Hydrophilicity 
Advanced Healthcare Materials  2014;4(5):695-701.
A new class of material resistant to bacterial attachment has been discovered that is formed from polyacrylates with hydrocarbon pendant groups. In this study, the relationship between the nature of the hydrocarbon moiety and resistance to bacteria is explored, comparing cyclic, aromatic, and linear chemical groups. A correlation is shown between bacterial attachment and a parameter derived from the partition coefficient and the number of rotatable bonds of the materials' pendant groups. This correlation is applicable to 86% of the hydrocarbon pendant moieties surveyed, quantitatively supporting the previous qualitative observation that bacteria are repelled from poly(meth)acrylates containing a hydrophilic ester group when the pendant group is both rigid and hydrophobic. This insight will help inform and predict the further development of polymers resistant to bacterial attachment.
doi:10.1002/adhm.201400648
PMCID: PMC4409840  PMID: 25491266
low-fouling; molecular descriptors; polymer microarrays; Pseudomonas aeruginosa; ion mass spectrometry
9.  Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells 
As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D.
doi:10.1038/mtm.2015.11
PMCID: PMC4445011  PMID: 26029722
10.  Comparison of four methods of analysis of lipoprotein particle subfractions for their association with angiographic progression of coronary artery disease 
Atherosclerosis  2014;233(2):713-720.
Background
Compare gradient gel electrophoresis (GGE), vertical auto profile (VAP-II), nuclear magnetic resonance spectroscopy (NMR), and ion mobility for their ability to relate low-(LDL), intermediate- (IDL), very-low-density (VLDL) and high-density lipoprotein (HDL) subfraction concentrations to atherosclerotic progression.
Methods and Results
Regression analyses of 136 patients who received baseline and follow-up coronary angiographies and subfraction measurements by all four methods in the HDL Atherosclerosis Treatment Study. Prior analyses have shown that the intervention primarily affected disease progression in proximal arteries with <30% stenoses at baseline.
Three-year increases in percent stenoses were consistently associated with higher on-study plasma concentrations of small, dense LDL as measured by GGE (LDLIIIb, P=10−6; LDLIVa, P=0.006; LDLIVb, P=0.02), VAP-II (LDL4, P=0.002), NMR (small LDL, P=0.001), and ion mobility (LDL IIb, P=0.04; LDLIIIa, P=0.002; LDLIIIb, P=0.0007; LDLIVa, P=0.05). Adjustment for triglycerides, HDL-cholesterol, and LDL-cholesterol failed to eliminate the statistical significance for on-study GGE estimated LDLIIIb (P=10−5) and LDLIVa (P=0.04); NMR-estimated small LDL (P=0.03); or ion mobility estimated large LDLIIIa (P=0.04) or LDLIIIb (P=0.02). All methods show that the effects were significantly greater for the on-study than the baseline small, dense LDL concentrations, thus establishing that the values concurrent to the progression of disease were responsible. The rate of disease progression was also related to individual VLDL, IDL, and HDL subclasses to differing extents among the various analytic methods.
Conclusion
Four methodologies confirm the association of small, dense LDL with greater coronary atherosclerosis progression, and GGE, NMR, and ion mobility confirm that the associations were independent of standard lipid measurements.
Clinical Trial Registration
clinicaltrials.gov/ct2/show/NCT00000553
doi:10.1016/j.atherosclerosis.2014.01.034
PMCID: PMC3990359  PMID: 24603218
Lipoprotein subfractions; low-density lipoproteins; very low density lipoproteins; high density lipoproteins; angiography; coronary artery disease; prevention
11.  Dose-Response Relationship between Exercise and Respiratory Disease Mortality 
Purpose
To assess prospectively the dose-response relationship between respiratory disease (ICD10: J1-99), pneumonia (ICD10: J12.0-18.9), and asperation pneumonia mortality (ICD10: J69) vs. baseline walking and running energy expenditure (MET-hours/d, 1 MET = 3.5 ml O2/kg/min).
Methods
Cox proportional hazard analyses of 109,352 runners and 40,798 walkers adjusted for age, sex, smoking, diet, alcohol, and education.
Results
There were 236 deaths with respiratory disease listed as the underlying cause, and 833 deaths that were respiratory disease related (entity axis diagnosis). Included among these were 79 deaths with pneumonia listed as the underlying cause and 316 pneumonia-related deaths, and 77 deaths due to aspiration pneumonia. There was no significant difference in the effect of running compared to walking (per MET-hours/d) on mortality, thus runners and walkers were combined for analysis. Respiratory disease mortality decreased 7.9% per MET-hours/d as the underlying cause (95%CI: 1.6% to 14.0%, P=0.01) and 7.3% for all respiratory disease-related deaths (95%CI: 4.2% to 10.4%, P=10-5). Pneumonia mortality decreased 13.1% per MET-hours/d as the underlying cause (95%CI: 2.6% to 23.2%, P=0.01) and 10.5% per MET-hours/d for all pneumonia-related deaths (95%CI: 5.4% to 15.5%, P=0.0001). The risk for aspiration pneumonia mortality also did not differ between running and walking, and decreased 19.9% per MET-hours/d run or walked (95%CI: 8.9% to 30.2%, P=0.0004). These results remained significant when additionally adjusted for BMI.
Conclusions
Higher doses of running and walking were associated with lower risk of respiratory disease, pneumonia, and aspiration pneumonia mortality in a dose-dependent manner, and the effects of running and walking appear equivalent. These effects appear to be independent of the effects of exercise on cardiovascular disease.
doi:10.1249/MSS.0000000000000142
PMCID: PMC4073613  PMID: 24002349
physical activity; pneumonia; prevention; cohort
12.  Managed Bumblebees Outperform Honeybees in Increasing Peach Fruit Set in China: Different Limiting Processes with Different Pollinators 
PLoS ONE  2015;10(3):e0121143.
Peach Prunus persica (L.) Batsch is self-compatible and largely self-fertile, but under greenhouse conditions pollinators must be introduced to achieve good fruit set and quality. Because little work has been done to assess the effectiveness of different pollinators on peach trees under greenhouse conditions, we studied ‘Okubo’ peach in greenhouse tunnels near Beijing between 2012 and 2014. We measured pollen deposition, pollen-tube growth rates, ovary development, and initial fruit set after the flowers were visited by either of two managed pollinators: bumblebees, Bombus patagiatus Nylander, and honeybees, Apis mellifera L. The results show that B. patagiatus is more effective than A. mellifera as a pollinator of peach in greenhouses because of differences in two processes. First, B. patagiatus deposits more pollen grains on peach stigmas than A. mellifera, both during a single visit and during a whole day of open pollination. Second, there are differences in the fertilization performance of the pollen deposited. Half of the flowers visited by B. patagiatus are fertilized 9–11 days after bee visits, while for flowers visited by A. mellifera, half are fertilized 13–15 days after bee visits. Consequently, fruit development is also accelerated by bumblebees, showing that the different pollinators have not only different pollination efficiency, but also influence the subsequent time course of fertilization and fruit set. Flowers visited by B. patagiatus show faster ovary growth and ultimately these flowers produce more fruit. Our work shows that pollinators may influence fruit production beyond the amount of pollen delivered. We show that managed indigenous bumblebees significantly outperform introduced honeybees in increasing peach initial fruit set under greenhouse conditions.
doi:10.1371/journal.pone.0121143
PMCID: PMC4370422  PMID: 25799170
13.  [18F]FDG-6-P as a novel in vivo tool for imaging staphylococcal infections 
EJNMMI Research  2015;5:13.
Background
Management of infection is a major clinical problem. Staphylococcus aureus is a Gram-positive bacterium which colonises approximately one third of the adult human population. Staphylococcal infections can be life-threatening and are frequently complicated by multi-antibiotic resistant strains including methicillin-resistant S. aureus (MRSA). Fluorodeoxyglucose ([18F]FDG) imaging has been used to identify infection sites; however, it is unable to distinguish between sterile inflammation and bacterial load. We have modified [18F]FDG by phosphorylation, producing [18F]FDG-6-P to facilitate specific uptake and accumulation by S. aureus through hexose phosphate transporters, which are not present in mammalian cell membranes. This approach leads to the specific uptake of the radiopharmaceutical into the bacteria and not the sites of sterile inflammation.
Methods
[18F]FDG-6-P was synthesised from [18F]FDG. Yield, purity and stability were confirmed by RP-HPLC and iTLC. The specificity of [18F]FDG-6-P for the bacterial universal hexose phosphate transporter (UHPT) was confirmed with S. aureus and mammalian cell assays in vitro. Whole body biodistribution and accumulation of [18F]FDG-6-P at the sites of bioluminescent staphylococcal infection were established in a murine foreign body infection model.
Results
In vitro validation assays demonstrated that [18F]FDG-6-P was stable and specifically transported into S. aureus but not mammalian cells. [18F]FDG-6-P was elevated at the sites of S. aureus infection in vivo compared to uninfected controls; however, the increase in signal was not significant and unexpectedly, the whole-body biodistribution of [18F]FDG-6-P was similar to that of [18F]FDG.
Conclusions
Despite conclusive in vitro validation, [18F]FDG-6-P did not behave as predicted in vivo. However at the site of known infection, [18F]FDG-6-P levels were elevated compared with uninfected controls, providing a higher signal-to-noise ratio. The bacterial UHPT can transport hexose phosphates other than glucose, and therefore alternative sugars may show differential biodistribution and provide a means for specific bacterial detection.
doi:10.1186/s13550-015-0095-1
PMCID: PMC4385282  PMID: 25853019
Pre-clinical; NanoPET-CT imaging; S. aureus; Infection diagnosis
14.  Mathematical modeling of positron emission tomography (PET) data to assess radiofluoride transport in living plants following petiolar administration 
Plant Methods  2015;11:18.
Background
Ion transport is a fundamental physiological process that can be studied non-invasively in living plants with radiotracer imaging methods. Fluoride is a known phytotoxic pollutant and understanding its transport in plants after leaf absorption is of interest to those in agricultural areas near industrial sources of airborne fluoride. Here we report the novel use of a commercial, high-resolution, animal positron emission tomography (PET) scanner to trace a bolus of [18F]fluoride administered via bisected petioles of Brassica oleracea, an established model species, to simulate whole plant uptake of atmospheric fluoride. This methodology allows for the first time mathematical compartmental modeling of fluoride transport in the living plant. Radiotracer kinetics in the stem were described with a single-parameter free- and trapped-compartment model and mean arrival times at different stem positions were calculated from the free-compartment time-activity curves.
Results
After initiation of administration at the bisected leaf stalk, [18F] radioactivity climbed for approximately 10 minutes followed by rapid washout from the stem and equilibration within leaves. Kinetic modeling of transport in the stem yielded a trapping rate of 1.5 +/− 0.3%/min (mean +/− s.d., n = 3), velocity of 2.2 +/− 1.1 cm/min, and trapping fraction of 0.8 +/− 0.5%/cm.
Conclusion
Quantitative assessment of physiologically meaningful transport parameters of fluoride in living plants is possible using standard positron emission tomography in combination with petiolar radiotracer administration. Movement of free fluoride was observed to be consistent with bulk flow in xylem, namely a rapid and linear change in position with respect to time. Trapping, likely in the apoplast, was observed. Future applications of the methods described here include studies of transport of other ions and molecules of interest in plant physiology.
Electronic supplementary material
The online version of this article (doi:10.1186/s13007-015-0061-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13007-015-0061-y
PMCID: PMC4359769  PMID: 25774208
Ion transport; Positron emission tomography; Radiotracer; Kinetic modeling; Brassica oleracea
15.  Conjugated linoleic acid prevents ovariectomy-induced bone loss in mice by modulating both osteoclastogenesis and osteoblastogenesis 
Lipids  2013;49(3):211-224.
Postmenopausal osteoporosis due to estrogen deficiency is associated with severe morbidity and mortality. Beneficial effects of conjugated linoleic acid (CLA) on bone mineral density (BMD) have been reported in mice, rats and humans, but the effect of long term CLA supplementation against ovariectomy-induced bone loss in mice and the mechanisms underlying this effect have not been studied yet. Eight weeks old ovariectomized (Ovx) and sham operated C57BL/6 mice were fed either a diet containing 0.5% safflower oil (SFO) or 0.5% CLA for 24 weeks to examine BMD, bone turn over markers and osteotropic factors. Bone marrow (BM) cells were cultured to determine the effect on inflammation, osteoclastogenesis, and osteoblastogenesis. SFO/Ovx mice had significantly lower femoral, tibial and lumbar BMD compared to SFO/Sham mice; whereas, no difference was found between CLA/OVX and CLA/Sham mice. CLA inhibited bone resorption markers whereas enhanced bone formation markers in Ovx mice as compared to SFO fed mice. RT-PCR and FACS analyses of splenocytes revealed that CLA inhibited pro-osteoclastogenic RANKL and stimulated decoy receptor of RANKL, OPG expression. CLA also inhibited pro-inflammatory cytokine and enhanced anti-inflammatory cytokine production of LPS-stimulated splenocytes and bone marrow cells. Furthermore, CLA inhibited osteoclast differentiation in BM and stimulated osteoblast differentiation in BM stromal cells as confirmed by TRAP and Alizarin Red staining, respectively. In conclusion, CLA may prevent postmenopausal bone loss not only by inhibiting excessive bone resorption due to estrogen deficiency but also by stimulating new bone formation. CLA might be a potential alternative therapy against osteoporotic bone loss.
doi:10.1007/s11745-013-3872-5
PMCID: PMC3947714  PMID: 24338525
Osteoporosis; ovariectomy; bone mineral density; osteoclastogenesis; osteoblastogenesis; inflammation
16.  CCN2 requires TGF-β signalling to regulate CCAAT/enhancer binding proteins and inhibit fat cell differentiation 
Introduction
Fat cell differentiation (FCD) potentiates adipose cell characteristics including lipid storage and insulin sensitivity. In vitro, we have demonstrated that CCN2, also known as connective tissue growth factor (CTGF), inhibits FCD in NIH3T3-L1 cells and in adipocytes isolated from mouse epididymal fat pads. The aim of this study was to determine if the CCN2 effect on FCD is dependent on TGF-β and TGF-β downstream pathway signalling.
Methods
NIH3T3-L1 cells were differentiated using standard methods with IBMX/Dex/Insulin. FCD at day 10 was confirmed by induced gene markers resistin and adiponectin and by lipid accumulation. Cells were treated at d0 with single dose active rhTGF-β1 (2 ng/mL), rhCCN2 (500 ng/mL) and/or TGF-β type 1 receptor blocker (SB431542, 5 μM). Early induction of FCD transcription factors: CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPAR-γ), were also determined.
Results
In an early time course from 2 h, single doses of rhTGF-β1 or rhCCN2 significantly inhibited by ~70 % the induction of C/EBP-β and -δ mRNA, and also nuclear protein levels otherwise seen during FCD, whereas only delayed effects on PPAR-γ, at 48 h, occurred. Furthermore, the CCN2 inhibition of FCD markers adiponectin and resistin and lipid accumulation by Oil red O stain were each prevented by TGF-β receptor blockade. Similar prevention was found using pan-specific anti-TGF-β neutralising antibody. CCN2 and TGF-β treatment each rapidly phosphorylated SMAD-3 signalling in early stages of FCD.
Conclusion
This work shows novel findings that CCN2 effects on FCD are both TGF-β and TGF-β pathway dependent and are related to early effects on C/EBPs.
doi:10.1007/s12079-014-0252-8
PMCID: PMC4414844  PMID: 25354561
CCN2; TGFBeta; Adipocyte; Signaling
17.  Genome Wide Association Study for Drought, Aflatoxin Resistance, and Important Agronomic Traits of Maize Hybrids in the Sub-Tropics 
PLoS ONE  2015;10(2):e0117737.
The primary maize (Zea mays L.) production areas are in temperate regions throughout the world and this is where most maize breeding is focused. Important but lower yielding maize growing regions such as the sub-tropics experience unique challenges, the greatest of which are drought stress and aflatoxin contamination. Here we used a diversity panel consisting of 346 maize inbred lines originating in temperate, sub-tropical and tropical areas testcrossed to stiff-stalk line Tx714 to investigate these traits. Testcross hybrids were evaluated under irrigated and non-irrigated trials for yield, plant height, ear height, days to anthesis, days to silking and other agronomic traits. Irrigated trials were also inoculated with Aspergillus flavus and evaluated for aflatoxin content. Diverse maize testcrosses out-yielded commercial checks in most trials, which indicated the potential for genetic diversity to improve sub-tropical breeding programs. To identify genomic regions associated with yield, aflatoxin resistance and other important agronomic traits, a genome wide association analysis was performed. Using 60,000 SNPs, this study found 10 quantitative trait variants for grain yield, plant and ear height, and flowering time after stringent multiple test corrections, and after fitting different models. Three of these variants explained 5–10% of the variation in grain yield under both water conditions. Multiple identified SNPs co-localized with previously reported QTL, which narrows the possible location of causal polymorphisms. Novel significant SNPs were also identified. This study demonstrated the potential to use genome wide association studies to identify major variants of quantitative and complex traits such as yield under drought that are still segregating between elite inbred lines.
doi:10.1371/journal.pone.0117737
PMCID: PMC4340625  PMID: 25714370
18.  Granulocyte-Macrophage Colony Stimulatory Factor Enhances the Pro-Inflammatory Response of Interferon-γ-Treated Macrophages to Pseudomonas aeruginosa Infection 
PLoS ONE  2015;10(2):e0117447.
Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ.
doi:10.1371/journal.pone.0117447
PMCID: PMC4338139  PMID: 25706389
19.  Genome Sequences of Two Pandoraea pnomenusa Isolates Recovered 11 Months Apart from a Cystic Fibrosis Patient 
Genome Announcements  2015;3(1):e01389-14.
Pandoraea is an emerging respiratory pathogen capable of causing chronic lung infections in people with cystic fibrosis (CF), but the clinical significance of this infection is ambiguous. We have sequenced and annotated the genomes of two multidrug-resistant Pandoraea pnomenusa isolates recovered 11 months apart from the same CF patient.
doi:10.1128/genomeA.01389-14
PMCID: PMC4319598  PMID: 25657265
20.  Src and Caveolin-1 reciprocally regulate metastasis via a common downstream signaling pathway in bladder cancer 
Cancer research  2010;71(3):832-841.
In bladder cancer, increased Caveolin-1 (Cav-1) expression and decreased Src expression and kinase activity correlate with tumor aggressiveness. Here, we investigate the clinical and functional significance if any, of this reciprocal expression in bladder cancer metastasis. We evaluated the ability of tumor Cav-1 and Src RNA and protein expression to predict outcome following cystectomy in 257 patients enrolled in two independent clinical studies. In both, high Cav-1 and low Src levels were associated with metastasis development. We overexpressed or depleted Cav-1 and Src protein levels in UMUC-3 and, RT4 human bladder cancer cells and evaluated the effect of this on actin stress fibers, migration using transwells and lung metastasis following tail vein inoculation. Cav-1 depletion or expression of active Src in metastatic UMUC-3 cells decreases actin stress fibers, cell migration and metastasis, while, Cav-1 overexpression or Src depletion increased the migration of non-metastatic RT4 cells. Biochemical studies indicated Cav-1 mediates these effects via its phosphorylated form (pY14), whereas Src effects are mediated through phosphorylation of p190RhoGAP and these pathways converge to reduce activity of RhoA, RhoC and Rho effector ROCK1. Treatment with a ROCK inhibitor reduced UMUC-3 lung metastasis in vivo, phenocopying the effect of Cav-1 depletion or expression of active Src. Src suppresses while Cav-1 promotes metastasis of bladder cancer through a pharmacologically tractable common downstream signaling pathway. Clinical evaluation of personalized therapy to suppress metastasis development based on Cav-1 and Src profiles appears warranted.
doi:10.1158/0008-5472.CAN-10-0730
PMCID: PMC4306590  PMID: 21148751
Caveolin-1; Src; Rho A; Rho C; metastasis; bladder neoplasms
21.  SELECTIVE ADVANTAGE OF RECOMBINATION IN EVOLVING PROTEIN POPULATIONS: A LATTICE MODEL STUDY 
Recent research has attempted to clarify the contributions of several mutational processes, such as substitutions or homologous recombination. Simplistic, tractable protein models, which determine the compact native structure phenotype from the sequence genotype, are well-suited to such studies. In this paper, we use a lattice-protein model to examine the effects of point mutation and homologous recombination on evolving populations of proteins. We find that while the majority of mutation and recombination events are neutral or deleterious, recombination is far more likely to be beneficial. This results in a faster increase in fitness during evolution, although the final fitness level is not significantly changed. This transient advantage provides an evolutionary advantage to subpopulations that undergo recombination, allowing fixation of recombination to occur in the population.
doi:10.1142/S0129183106008959
PMCID: PMC4249953  PMID: 25473139
homologous protein recombination; introns; lattice-protein
22.  DHA is a more potent inhibitor of breast cancer metastasis to bone and related osteolysis than EPA 
Breast cancer research and treatment  2013;141(3):10.1007/s10549-013-2703-y.
Breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Attenuation of breast cancer metastasis to bone and associated osteolysis by fish oil (FO), rich in EPA and DHA, has been demonstrated previously. However, it was not known whether EPA and DHA differentially or similarly affect breast cancer bone metastasis and associated osteolysis. In vitro culture of parental and luciferase gene encoded MDA-MB-231 human breast cancer cell lines treated with EPA and DHA revealed that DHA inhibits proliferation and invasion of breast cancer cells more potently than EPA. Intra-cardiac injection of parental and luciferase gene encoded MDA-MB-231 cells to athymic NCr nu/nu mice demonstrated that DHA treated mice had significantly less breast cancer cell burden in bone, and also significantly less osteolytic lesions than EPA treated mice. In vivo cell migration assay as measured by luciferase intensity revealed that DHA attenuated cell migration specifically to the bone. Moreover, the DHA treated group showed reduced levels of CD44 and TRAP positive area in bone compared to EPA treated group. Breast cancer cell burden and osteolytic lesions were also examined in intra-tibially breast cancer cell injected mice and found less breast cancer cell growth and associated osteolysis in DHA treated mice as compared to EPA treated mice. Finally, doxorubicin resistant MCF-7 (MCF-7dox) human breast cancer cell line was used to examine if DHA can improve sensitization of MCF-7dox cells to doxorubicin. DHA improved the inhibitory effect of doxorubicin on proliferation and invasion of MCF-7dox cells. Interestingly, drug resistance gene P-gp was also down-regulated in DHA plus doxorubicin treated cells. In conclusion, DHA attenuates breast cancer bone metastasis and associated osteolysis more potently than EPA, possibly by inhibiting migration of breast cancer cell to the bone as well as by inhibiting osteoclastic bone resorption.
doi:10.1007/s10549-013-2703-y
PMCID: PMC3839302  PMID: 24062211
Breast cancer; bone metastasis; omega-3 fatty acids; docosahexaenoic acid; osteolysis
23.  Walking and running produce similar reductions in cause-specific disease mortality in hypertensives 
Hypertension  2013;62(3):485-491.
To test prospectively in hypertensives whether moderate and vigorous exercise produce equivalent reductions in mortality, Cox-proportional hazard analyses were applied to energy expenditure (metabolic equivalents hours/day, METh/d) in 6,973 walkers and 3,907 runners who used hypertensive medications at baseline. 1121 died during 10.2-year follow-up: 695 cardiovascular disease (CVD, ICD10 I00-99, 465 underlying cause, 230 contributing cause), 124 cerebrovascular disease, 353 ischemic heart disease (ICD10 I20-25, 257 underlying, 96 contributing), 122 heart failure (ICD10 I50, 24 underlying, 98 contributing), and 260 dysrhythmias (ICD10 I46-49, 24 underlying, 236 contributing). Relative to <1.07 METh/d, running or walking 1.8-3.6 METh/d produced significantly lower all-cause (29% reduction, 95%CI: 17% to 39%, P=0.0001), CVD (34% reduction, 95%CI: 20% to 46%, P=0.0001), cerebrovascular disease (55% reduction, 95%CI: 27% to 73%, P=0.001), dysrhythmia (47% reduction, 95%CI: 27% to 62%, P=0.0001), and heart failure mortality (51% reduction, 95%CI: 21% to 70%, P=0.003), as did ≥3.6 METh/d with all-cause (22% reduction, 95%CI: 6% to 35%, P=0.005), CVD (36% reduction, 95%CI: 19% to 50%, P=0.0002), cerebrovascular disease (47% reduction, 95%CI: 6% to 71%, P=0.03), and dysrhythmia mortality (43% reduction, 95%CI: 16% to 62%, P=0.004). Diabetes and chronic kidney disease mortality also decreased significantly with METh/d. All results remained significant when BMI adjusted. Merely meeting guideline levels (1.07 to 1.8 METh/d) did not significantly reduced mortality. The dose-response was significantly nonlinear for all endpoints except diabetes, and cerebrovascular and chronic kidney disease. Results did not differ between running and walking. Thus, walking and running produce similar reductions in mortality in hypertensives.
doi:10.1161/HYPERTENSIONAHA.113.01608
PMCID: PMC4090350  PMID: 23940195
Hypertension; Exercise; Cardiovascular diseases; Diabetes Mellitus; Type 2; Renal Insufficiency; chronic
24.  Beyond Punnett Squares: Student Word Association and Explanations of Phenotypic Variation through an Integrative Quantitative Genetics Unit Investigating Anthocyanin Inheritance and Expression in Brassica rapa Fast Plants 
CBE Life Sciences Education  2014;13(3):410-424.
This study explores shifts in student word association and explanations of phenotypic variation through an integrative quantitative genetics unit using Brassica rapa Fast Plants.
Genetics instruction in introductory biology is often confined to Mendelian genetics and avoids the complexities of variation in quantitative traits. Given the driving question “What determines variation in phenotype (Pv)? (Pv=Genotypic variation Gv + environmental variation Ev),” we developed a 4-wk unit for an inquiry-based laboratory course focused on the inheritance and expression of a quantitative trait in varying environments. We utilized Brassica rapa Fast Plants as a model organism to study variation in the phenotype anthocyanin pigment intensity. As an initial curriculum assessment, we used free word association to examine students’ cognitive structures before and after the unit and explanations in students’ final research posters with particular focus on variation (Pv = Gv + Ev). Comparison of pre- and postunit word frequency revealed a shift in words and a pattern of co-occurring concepts indicative of change in cognitive structure, with particular focus on “variation” as a proposed threshold concept and primary goal for students’ explanations. Given review of 53 posters, we found ∼50% of students capable of intermediate to high-level explanations combining both Gv and Ev influence on expression of anthocyanin intensity (Pv). While far from “plug and play,” this conceptually rich, inquiry-based unit holds promise for effective integration of quantitative and Mendelian genetics.
doi:10.1187/cbe.13-12-0232
PMCID: PMC4152203  PMID: 25185225
25.  Localized Flux Maxima of Arsenic, Lead, and Iron around Root Apices in Flooded Lowland Rice 
Environmental Science & Technology  2014;48(15):8498-8506.
In wetland-adapted plants, such as rice, it is typically root apexes, sites of rapid entry for water/nutrients, where radial oxygen losses (ROLs) are highest. Nutrient/toxic metal uptake therefore largely occurs through oxidized zones and pH microgradients. However, the processes controlling the acquisition of trace elements in rice have been difficult to explore experimentally because of a lack of techniques for simultaneously measuring labile trace elements and O2/pH. Here, we use new diffusive gradients in thin films (DGT)/planar optode sandwich sensors deployed in situ on rice roots to demonstrate a new geochemical niche of greatly enhanced As, Pb, and Fe(II) mobilization into solution immediately adjacent to the root tips characterized by O2 enrichment and low pH. Fe(II) mobilization was congruent to that of the peripheral edge of the aerobic root zone, demonstrating that the Fe(II) mobilization maximum only developed in a narrow O2 range as the oxidation front penetrates the reducing soil. The Fe flux to the DGT resin at the root apexes was 3-fold higher than the anaerobic bulk soil and 27 times greater than the aerobic rooting zone. These results provide new evidence for the importance of coupled diffusion and oxidation of Fe in modulating trace metal solubilization, dispersion, and plant uptake.
doi:10.1021/es501127k
PMCID: PMC4124062  PMID: 24967508

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