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1.  Lentivirus-Activated T Regulatory Cells Suppress T Helper Cell Interleukin-2 Production by Inhibiting Nuclear Factor of Activated T Cells 2 Binding to the Interleukin-2 Promoter 
Abstract
Using the feline immunodeficiency virus (FIV) model for AIDS lentivirus infection, we previously demonstrated that Treg cells from FIV-infected cats up-regulate membrane-associated tumor growth factor beta (mTGF-ß) during the course of infection and that activated T lymphocytes up-regulate TGF-ß receptor II (TGF-ßRII) during the course of infection. Furthermore, we have demonstrated that autologous coculture of Tregs with Th cells from FIV-infected cats leads to suppression of interleukin (IL)-2 production and loss of proliferation in a TGF-ß-dependent fashion. Nuclear factor of activated T cells (NFAT) 2 has been identified as integral to effector Th cell maturation and function by promoting IL-2 transcription. Therefore, we questioned whether NFAT2 expression might be altered by TGF-β signaling. Feline NFAT2 exon sequences were identified based upon sequence homology to human and murine NFAT2. Following stimulation, IL-2 and NFAT2 mRNA levels were similarly increased in both FIV− and FIV+ cats. Activated CD4+CD25− cells from both FIV− and FIV+ cats cocultured with autologous CD4+CD25+ cells or treated with TGF-β demonstrated decreased IL-2 production; however, NFAT2 mRNA levels were unaffected. Although NFAT2 mRNA levels were unaffected, chromatin immunoprecipitation (ChIP) for NFAT2 indicated decreased NFAT2 binding at the IL-2 promoter in suppressed Th cells. These data suggest that TGF-β-mediated Treg cell suppression of IL-2 transcription is modulated through alterations in NFAT2 binding to the IL-2 promoter.
doi:10.1089/aid.2013.0062
PMCID: PMC3887409  PMID: 23924068
2.  Anti-tumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding 
Journal of medicinal chemistry  2012;55(11):5121-5129.
Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, 15N-1H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin’s carbohydrate binding site, and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of anti-tumor calixarene compound 0118.
doi:10.1021/jm300014q
PMCID: PMC4242090  PMID: 22575017
NMR; protein; lectin; glycan; galactose
3.  Change in dietary intake of adults with intermittent claudication undergoing a supervised exercise program and compared to matched controls 
Nutrition Journal  2014;13(1):100.
Background
Presence of numerous diet responsive comorbidities and high atherosclerotic burden among adults with intermittent claudication demands attention is given to diet in an effort to delay progression of peripheral artery disease. The aim of this study was to compare diet of adults with intermittent claudication: (a) against dietary recommendations; (b) following 12 weeks of supervised exercise training; and (c) against non-peripheral artery disease controls.
Methods
Diet was assessed using a food frequency questionnaire pre and post supervised exercise training. Pre-exercise diet was compared against Suggested Dietary Targets and against non-peripheral artery disease controls matched for gender, age and body weight. Pre-exercise diet was also compared against post-exercise diet.
Results
Pre-exercise 25/31 participants, 5/31 participants, 16/31 participants and 4/31 participants achieved recommendations for protein, carbohydrate, total fat and saturated fat respectively. Few achieved recommended intakes for fibre (3/31 participants), cholesterol (8/31 participants), folate (11/31 participants), potassium (1/31 participants), sodium (4/31 participants), retinol equivalents (1/31 participants) and vitamin C (3/31 participants). There were no differences observed between participants compared to controls in achievement of recommendations. Post-exercise, marginally more participants were able to achieve targets for cholesterol, sodium and vitamin C but not for any other nutrients.
Conclusions
Despite evidence to support benefits of dietary modification in risk reduction of peripheral artery disease, adults with intermittent claudication continue to consume poor diets. Research is required to determine whether dietary changes can be achieved with greater attention to nutrition counselling and the impact assessed in terms of delayed disease progression and long term health outcomes.
Trial Registration
ClinicalTrials.gov: NCT01871779.
doi:10.1186/1475-2891-13-100
PMCID: PMC4210553  PMID: 25316347
Peripheral arterial disease; Diet; Nutrition; Supervised exercise; Claudication
4.  Associations Between Change in Sleep Duration and Inflammation: Findings on C-reactive Protein and Interleukin 6 in the Whitehall II Study 
American Journal of Epidemiology  2013;178(6):956-961.
Cross-sectional evidence suggests associations between sleep duration and levels of the inflammatory markers, C-reactive protein and interleukin-6. This longitudinal study uses data from the London-based Whitehall II study to examine whether changes in sleep duration are associated with average levels of inflammation from 2 measures 5 years apart. Sleep duration (≤5, 6, 7, 8, ≥9 hours on an average week night) was assessed in 5,003 middle-aged women and men in 1991/1994 and 1997/1999. Fasting levels of C-reactive protein and interleukin-6 were measured in 1997/1999 and 2002/2004. Cross-sectional analyses indicated that shorter sleep is associated with higher levels of inflammatory markers. Longitudinal analyses showed that each hour per night decrease in sleep duration between 1991/1994 and 1997/1999 was associated with higher levels of C-reactive protein (8.1%) and interleukin-6 (4.5%) averaged across measures in 1997/1999 and 2002/2004. Adjustment for longstanding illness and major cardiometabolic risk factors indicated that disease processes may partially underlie these associations. An increase in sleep duration was not associated with average levels of inflammatory markers. These results suggest that both short sleep and reductions in sleep are associated with average levels of inflammation over a 5-year period.
doi:10.1093/aje/kwt072
PMCID: PMC3817449  PMID: 23801012
change in sleep duration; C-reactive protein; inflammatory markers; interleukin-6; sleep duration
5.  Structural basis of nSH2 regulation and lipid binding in PI3Kα 
Oncotarget  2014;5(14):5198-5208.
We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
PMCID: PMC4170646  PMID: 25105564
PIK3R1; p85; PIK3CA; PI3K; PIP2; PIP3
6.  Cross-Sectional Study of Sleep Quantity and Quality and Amnestic and Non-Amnestic Cognitive Function in an Ageing Population: The English Longitudinal Study of Ageing (ELSA) 
PLoS ONE  2014;9(6):e100991.
Background
The aim was to investigate the association between sleep disturbances and cognitive function in younger and older individuals from an ageing population.
Methods
3,968 male and 4,821 female white participants, aged 50 years and over, from the English Longitudinal Study of Ageing (ELSA) were studied. Information on sleep quality and quantity as well as both amnestic (memory, ACF) and non-amnestic (non-memory, nACF) function was available at Wave 4 (2008). Analysis of covariance was used to evaluate the relationship between sleep and cognitive function.
Results
After adjustment for multiple confounders in the younger group (50–64 years) duration of sleep explained 15.2% of the variance in ACF (p = 0.003) and 20.6% of nACF (p = 0.010). In the older group (65+ years) the estimates were 21.3% (p<0.001) and 25.6% (p<0.001), respectively. For sleep quality, there was a statistically significant association between sleep quality and both ACF (p<0.001) and nACF (p<0.001) in the older age group, but not in the younger age group (p = 0.586 and p = 0.373, respectively; interaction between age and sleep quality in the study sample including both age groups: p<0.001 for ACF and p = 0.018 for nACF). Sleep quality explained between 15.1% and 25.5% of the variance in cognition. The interaction with age was independent of duration of sleep. At any level of sleep duration there was a steeper association between sleep quality and ACF in the older than the younger group.
Conclusions
The associations between sleep disturbances and cognitive function vary between younger and older adults. Prospective studies will determine the temporal relationships between sleep disturbances and changes in cognition in different age groups.
doi:10.1371/journal.pone.0100991
PMCID: PMC4072772  PMID: 24968354
7.  Pyridinylquinazolines Selectively Inhibit Human Methionine Aminopeptidase-1 in Cells 
Journal of medicinal chemistry  2013;56(10):3996-4016.
Methionine aminopeptidases (MetAPs) which remove the initiator methionine from nascent peptides are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1–4). But all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1–4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.
doi:10.1021/jm400227z
PMCID: PMC3714011  PMID: 23634668
8.  Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity 
Glycobiology  2013;23(5):508-523.
The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of 15N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 103 M−1 and K2 = 3.4 ± 0.8 × 103 M−1. Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.
doi:10.1093/glycob/cwt005
PMCID: PMC3608354  PMID: 23376190
circular dichroism; FRET; galectin; molecular dynamics; NMR
9.  CD8+ clonality is associated with prolonged acute plasma viremia and altered mRNA cytokine profiles during the course of Feline Immunodeficiency Virus infection 
Acute lentiviral infection is characterized by early CD8+ cytotoxic T cell (CTL) activity and a subsequent decline in plasma viremia. However, CD8+ lymphocytes fail to eliminate the virus and a progressive T cell immune dysfunction develops during the course of chronic lentiviral infection. To further define this CD8+ immune dysfunction we utilized PARR (PCR for Antigen Receptor Rearrangements), a technique which measures clonally expanded lymphocyte populations by comparison of highly conserved T cell receptor (TCR) regions to identify the prevalence of clonal CD8+ T cells following FIV infection. We then compared phenotype, mRNA profiles, CD8+ proliferation and plasma viremia during acute and chronic infection for PARR positive (PARR+) and PARR negative (PARR−) Feline Immunodeficiency virus (FIV) infected cats. We demonstrated that approximately forty percent of the FIV+ cats examined exhibit CD8+ clonality compared to none of the FIV− control cats. There were no phenotypic differences between PARR+ and PARR− CD8+ lymphocytes from FIV+ cats but retrospective analysis of plasma viremia over the course of infection revealed a delayed peak in plasma viremia and a decline in lymphocyte counts were observed in the PARR+ group during acute infection. CD8+ lymphocytes isolated from chronically infected PARR− cats exhibited significantly higher mRNA expression of IFN-γ and IL-2 following mitogenic stimulation when compared to PARR+ CD8+ lymphocytes. These data suggest that clonal CD8+ expansion may be related to impaired control of acute viremia and less effective CD8+ anti-viral function. Using PARR to assess changes in CD8+ clonality during the progression from acute to chronic FIV infection may help to better characterize the factors which contribute to CD8+ anergy and lentiviral persistence.
doi:10.1016/j.vetimm.2012.12.005
PMCID: PMC3595358  PMID: 23332729
FIV; AIDS; CTL; CD8+ clonality; PARR
10.  Feline Glycoprotein A Repetitions Predominant Anchors Transforming Growth Factor Beta on the Surface of Activated CD4+CD25+ Regulatory T Cells and Mediates AIDS Lentivirus-Induced T Cell Immunodeficiency 
Abstract
Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP+TGFb+ Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP+ Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb+ Treg-mediated T cell immune suppression during lentivirus infection.
doi:10.1089/aid.2012.0322
PMCID: PMC3607965  PMID: 23373523
11.  L-Aminoacyl-triazine derivatives are isoform-selective PI3Kβ inhibitors that target non-conserved Asp862 of PI3Kβ 
ACS medicinal chemistry letters  2013;4(2):206-210.
A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor ZSTK474 were identified as potent and isoform selective inhibitors of PI3Kβ. The compounds showed selectivity based upon stereochemistry with L-amino acyl derivatives preferring PI3Kβ while their D-congeners favoured PI3Kδ. The mechanistic basis of this inhibition was studied using site-directed mutants. One Asp residue, D862 was identified as a critical participant in binding to the PI3Kβ-selective inhibitors distinguishing this class from other reported PI3Kβ-selective inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells.
doi:10.1021/ml300336j
PMCID: PMC3688631  PMID: 23795239
PI3 kinase; p110β; ZSTK474; cancer
12.  l-Aminoacyl-triazine Derivatives Are Isoform-Selective PI3Kβ Inhibitors That Target Nonconserved Asp862 of PI3Kβ 
ACS Medicinal Chemistry Letters  2012;4(2):206-210.
A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor ZSTK474 were identified as potent and isoform-selective inhibitors of PI3Kβ. The compounds showed selectivity based upon stereochemistry with l-amino acyl derivatives preferring PI3Kβ, while their d-congeners favored PI3Kδ. The mechanistic basis of this inhibition was studied using site-directed mutants. One Asp residue, D862, was identified as a critical participant in binding to the PI3Kβ-selective inhibitors, distinguishing this class from other reported PI3Kβ-selective inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells.
doi:10.1021/ml300336j
PMCID: PMC3688631  PMID: 23795239
PI3 kinase; p110β; ZSTK474; cancer
13.  Infection with Feline Immunodeficiency Virus Directly Activates CD4+ CD25+ T Regulatory Cells 
Journal of Virology  2013;87(16):9373-9378.
Lentivirus infection activates CD4+ CD25+ T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor β (TGF-β). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.
doi:10.1128/JVI.00996-13
PMCID: PMC3754053  PMID: 23760252
14.  CD4+CD25+ T regulatory cells activated during feline immunodeficiency virus infection convert T helper cells into functional suppressors through a membrane-bound TGFβ / GARP-mediated mechanism 
Virology Journal  2014;11:7.
Background
We and others have previously reported that cell membrane-bound TGFβ (mTGFβ) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFβ to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFβ+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection.
Results
Here, we report that Treg cells from FIV+ cats express GARP and mTGFβ and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFβ or anti-GARP treatment of Treg cells or by anti-TGFβRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFβ/TGFβRII signaling and that cell-surface GARP plays a major role in this process.
Conclusions
These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.
doi:10.1186/1743-422X-11-7
PMCID: PMC3902059  PMID: 24438223
FIV; HIV; AIDS; Lentivirus; Treg cells; mTGFβ; GARP
15.  Investigation of the effects of a high fish diet on inflammatory cytokines, blood pressure, and lipids in healthy older Australians 
Food & Nutrition Research  2014;58:10.3402/fnr.v58.20369.
Background
Aging is a condition of chronic inflammation. In healthy Australians ≥64 years, the primary aim was to determine whether four servings/week of mixed fish (FISH) improves serum cytokines (i.e. C-reactive protein (CRP), IL-1, IL-6, TNF-α) compared to a diet low in fish (<1 serving/week, CONTROL); the secondary aims were to assess the effect of the diet on blood pressure and serum lipids (TC, HDL-C, TG, calculated LDL-C).
Methods
An 8-week randomized, parallel study, stratified by CRP (<3 mg/L vs. ≥3 mg/L) on entry to the study. Compliance was measured using 3-day weighed food records in weeks 1 and 7 of the study. A 12-h fasting blood sample was taken at baseline and 8-weeks for erythrocyte fatty acids as confirmation of compliance, and measurement of serum cytokines and lipids. Blood pressure was measured at both time points.
Results
Eighty participants completed the study (mean (SD) age: 69.6 (5.8) years). During week 1 of the study, mean ± SEM daily dietary intake of very long chain omega-3 polyunsaturated fatty acids (VLCN n-3 PUFA) in FISH vs. CONTROL was 1,676±129 mg vs. 27±5 mg (p<0.001). Mean (SD) gram intake of study fish and meat was 121 (45) g and 123 (78) g, for those allocated to FISH and CONTROL, respectively. Mean ± SEM percentage VLCN n-3 PUFA in erythrocytes at 8-weeks was higher in those allocated to FISH vs. CONTROL (10.2±0.2% vs. 8.2±0.3%, p<0.001). There was no between-group difference in CRP (n=80), IL-1β (n=33) or IL-6 (n=21) concentrations, blood pressure, or lipids, at 8-weeks.
Conclusions
Eight weeks consumption of four servings/week fish did not affect serum cytokine concentrations, blood pressure or lipids compared to a diet low in fish. In healthy older adults with low inflammatory burden, our results do not support that short-term consumption of mixed fish has a beneficial effect on selected cardiovascular biomarkers.
doi:10.3402/fnr.v58.20369
PMCID: PMC3895208  PMID: 24454276
older adults; fish; C-reactive protein; omega-3 long chain fatty acids; blood pressure; lipids
16.  T-Cell Factor/β-Catenin Activity is Suppressed Normally in Two Independent Models of Autosomal Dominant Polycystic Kidney Disease 
Kidney international  2011;80(2):10.1038/ki.2011.56.
During murine kidney development, canonical WNT signalling is highly active in the renal tubules until about embryonic day E16-E18 when β-catenin transcriptional activity becomes progressively restricted to the nephrogenic zone. Several in vitro studies have found a link between cilial signalling and β-catenin regulation. The cilial protein genes PKD1 and PKD2 are known to be mutated in autosomal dominant polycystic kidney disease and previous studies proposed that these mutations could lead to a failure to suppress canonical WNT signalling activity; aberrant activity might then contribute to the cystic phenotype. However, in this study, we show that suppression of canonical WNT activity, defined by a TCF/β-catenin-lacZ reporter, is normal in two independent models of polycystic kidney disease. We crossed a TCF/β-catenin-lacZ reporter mouse with mice Pkd1 or Pkd2 mutations and found that there was no β-galactosidase staining in cells lining the renal cysts. This suggests that excessive β-catenin transcriptional activity may not contribute to cystogenesis in these models of autosomal dominant polycystic kidney disease.
doi:10.1038/ki.2011.56
PMCID: PMC3879791  PMID: 21389971
17.  Theoretical dietary modelling of Australian seafood species to meet long-chain omega 3 fatty acid dietary recommendations 
Food & Nutrition Research  2013;57:10.3402/fnr.v57i0.20737.
Background
Several agencies recommend seafood to be consumed 2–3 times per week. In Australia, there is a lack of nutrient composition data for seafood species and it is not known whether including different seafood species in a diet would provide sufficient long-chain omega 3 fatty acids (LC n–3 PUFA) to meet various national recommendations.
Objective
To utilise recent nutrient composition data for major Australian seafood groups (n=24) with the addition of two tuna options (total n=26) to: (1) determine whether including these species into a diet based on the Australian Guide to Healthy Eating (AGHE) will achieve LC n–3 PUFA recommendations [Adequate Intake (AI: 160 mg/d men, 90 mg/d women)], Suggested Dietary Target (SDT), 500 mg/d Heart Foundation (HF) recommendation and (2) determine the weekly number of servings of seafood to meet recommendations using either lower fat (n=23, <10% total fat) or higher fat (n=3, ≥10% total fat) seafood.
Design
Two simulation models incorporated all 26 species of seafood or only lower fat seafood into a diet based on the AGHE. Two further models identified the number of servings of lower or higher fat seafood required to meet recommendations.
Results
Including 2 and 3 servings/week of any seafood would enable 89% of women and 66% of men to meet the AI. Including only lower fat seafood would enable 83% of women and 47% of men to meet the AI. Half a serving/week of higher fat seafood would enable 100% of men and women to meet the AI.
Conclusions
Including the recommended 2–3 servings of seafood/week requires at least some higher fat seafood to be consumed in order for most men and women to meet the AI. Further messages and nutrition resources are needed which provide options on how to increase intake of LC n–3 PUFA, specifically through consumption of the higher fat seafood.
doi:10.3402/fnr.v57i0.20737
PMCID: PMC3813826  PMID: 24179469
seafood modelling; older adults; long-chain omega 3 fatty acids; seafood recommendations; Australia
18.  Now What? Effects of On-Site Assessment on Treatment Entry After Perinatal Depression Screening 
Journal of Women's Health  2012;21(10):1046-1052.
Abstract
Background
Depression is a frequent accompaniment of the perinatal period. Although screening improves detection of perinatal depression, it does not in itself improve mental health treatment entry and, therefore, does not improve outcomes. This study addresses the feasibility of incorporating diagnostic assessment for depression directly into perinatal care visits and the influence of doing so on entry into mental health treatment.
Methods
The Perinatal Depression Management Program was implemented in an urban community health center serving a predominantly Hispanic population. The Patient Health Questionnaire (PHQ-9) was administered during perinatal visits. Positive screens (scores ≥10) were followed within the same visit by brief diagnostic assessment and engagement strategies. Chart review was conducted to compare rates of screening, assessment, and treatment entry during a 3-month baseline period before implementation of the intervention (n=141) with a 1-year period after implementation of the intervention (n=400).
Results
Before the intervention, 65.2% of patients completed a PHQ-9, and 10% of patients with positive screens received on-site assessment. None of the patients with identified perinatal depression entered treatment. After model implementation, significantly more (93.5%) completed a PHQ-9, and of patients with positive screens, 84.8% received an on-site assessment. Among patients diagnosed with major depression and offered treatment, 90% entered treatment.
Conclusions
It is feasible to implement diagnostic assessment for depression within perinatal clinic visits. Doing so may substantially increase entry into mental health treatment for women with perinatal major depression while reducing unnecessary mental health referral of patients with false positive screens.
doi:10.1089/jwh.2012.3641
PMCID: PMC3466908  PMID: 22994985
19.  Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in Chlamydomonas reinhardtii 
PLoS Genetics  2013;9(9):e1003841.
Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.
Author Summary
Whole genome sequencing is a powerful tool to detect changes in genomic DNA. However, how to identify a causative mutation from over 20,000 changes remains a big challenge. For the unicellular green alga Chlamydomonas, we built a library that consists of over 2 million changes from 16 strains. A comparison of changes found in a mutant strain with a mating defect, imp3, to 15 other strains, leads to the identification of a three amino acid deletion in the catalytic subunit of a protein phosphatase 2A (PP2A3). The mating defect of imp3 is rescued by an HA-tagged PP2A3 gene. Introduction of the HA-tagged PP2A3 gene with various mutations in these three amino acids reveals that they play a key role in stabilizing and ensuring the proper localization of PP2A3. The ubiquitous enzyme PP2A is involved in diverse cellular processes. Our discovery that PP2A3 is involved in the Chlamydomonas mating signaling pathway, which also contains the polycystin2 homolog (PKD2), makes Chlamydomonas mating an excellent model to study ciliary/flagellar signaling. Since both PP2A and PKD2 play important roles in human health, further investigation of the relationship between these two proteins in Chlamydomonas will facilitate better understanding of their functions.
doi:10.1371/journal.pgen.1003841
PMCID: PMC3784568  PMID: 24086163
20.  Development and relative validity of a new field instrument for detection of geriatric cachexia: preliminary analysis in hip fracture patients 
Background
Geriatric cachexia is distinct from other age-related muscle wasting syndromes; however, detection and therefore treatment is challenging without the availability of valid instruments suitable for application in the clinical setting. This study assessed the sensitivity and specificity of a newly developed screening instrument utilising portable assessments against previously defined and commonly accepted diagnostic criteria for detection of geriatric cachexia.
Methods
Cross-sectional analyses from 71 older adults’ post-surgical fixation for hip fracture were performed. The diagnostic criteria required measures of appendicular skeletal muscle index derived from dual-energy X-ray absorptiometry and anorexia assessed by ≤70 % of estimated energy requirements. These assessments were replaced with mid-upper arm muscle circumference and the Simplified Nutritional Appetite Questionnaire, respectively, to create a field instrument suitable for screening geriatric cachexia. Sensitivity, specificity and positive and negative predictive values were calculated.
Results
The current diagnostic algorithm identified few patients as cachectic (4/71; 5.6 %). The sensitivity and specificity of the geriatric cachexia screening tool was 75 and 97 %, respectively. The screening tool had a positive predictive value of 60 % and a negative predictive value of 99 %.
Conclusions
Given the unexpected prevalence of cachexia in such a vulnerable group, these results may suggest problems in operationalising of the consensus definition and diagnostic criteria. Although the application of a newly developed screening tool using portable field measures looks promising, the authors recommend additional research to identify the prevalence of geriatric cachexia, which captures all diagnostic criteria from the consensus definition. Future investigation may then be positioned to explore the predictive validity of screening tools using portable field measures, which potentially achieve higher sensitivity.
doi:10.1007/s13539-013-0108-8
PMCID: PMC3774920  PMID: 23686412
Cachexia; Older adults; Hip fracture; Validity; Reliability
21.  Pathways of introduction of the invasive aquatic plant Cabomba caroliniana 
Ecology and Evolution  2013;3(6):1427-1439.
The pathway and frequency of species' introductions can affect the extent, impact, and management of biological invasions. Here, we examine the pathway of introduction of the aquatic plant Cabomba caroliniana (fanwort) into Canada and the northern United States using plastid DNA sequence (intergenic spacers atpF-atpH, trnH-psbA, and trnL-trnF) and DNA content analyses. We test the hypothesis that the spread of fanwort is a result of commercial trade by comparing a Canadian population (Kasshabog Lake, ON) to native populations from southern U.S., introduced populations in northern U.S., and plants from commercial retailers. Thirteen plastid haplotypes were identified throughout North America, including one dominant haplotype, which was present in all C. caroliniana populations. Several rare haplotypes were used to infer shared colonization history. In particular, the Canadian population shared two rare alleles with a population from Massachusetts, suggesting range expansion of C. caroliniana from the northern U.S. However, the possibility of a commercial introduction cannot be excluded, as common alleles were shared between the Canadian population and both commercial and southern U.S. sources. Variation in C. caroliniana genome size was bimodal and populations were classified into “high” and “low” categories. The Canadian population had DNA contents similar to several northern U.S. populations (low DNA content). This may provide additional support for range expansion from these introduced populations rather than from commercial sources or populations in the southern U.S., which had high DNA content.
doi:10.1002/ece3.530
PMCID: PMC3686180  PMID: 23789056
Fanwort; genome size; haplotype network; invasive species; molecular variation; polyploidy
22.  Fish oil administration in older adults: is there potential for adverse events? A systematic review of the literature 
BMC Geriatrics  2013;13:41.
Background
Omega-3 (n-3) fatty acid supplementation is becoming increasingly popular. However given its antithrombotic properties the potential for severe adverse events (SAE) such as bleeding has safety implications, particularly in an older adult population. A systematic review of randomized control trials (RCT) was conducted to explore the potential for SAE and non-severe adverse events (non-SAE) associated with n-3 supplementation in older adults.
Methods
A comprehensive search strategy using Medline and a variety of other electronic sources was conducted. Studies investigating the oral administration of n-3 fish oil containing eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or both against a placebo were sourced. The primary outcome of interest included reported SAE associated with n-3 supplementation. Chi-square analyses were conducted on the pooled aggregate of AEs.
Results
Of the 398 citations initially retrieved, a total of 10 studies involving 994 older adults aged ≥60 years were included in the review. Daily fish oil doses ranged from 0.03 g to 1.86 g EPA and/or DHA with study durations ranging from 6 to 52 weeks. No SAE were reported and there were no significant differences in the total AE rate between groups (n-3 intervention group: 53/540; 9.8%; placebo group: 28/454; 6.2%; p = 0.07). Non-SAE relating to gastrointestinal (GI) disturbances were the most commonly reported however there was no significant increase in the proportion of GI disturbances reported in participants randomized to the n-3 intervention (n-3 intervention group: 42/540 (7.8%); placebo group: 24/454 (5.3%); p = 0.18).
Conclusions
The potential for AEs appear mild-moderate at worst and are unlikely to be of clinical significance. The use of n-3 fatty acids and the potential for SAE should however be further researched to investigate whether this evidence is consistent at higher doses and in other populations. These results also highlight that well-documented data outlining the potential for SAE following n-3 supplementation are limited nor adequately reported to draw definitive conclusions concerning the safety associated with n-3 supplementation. A more rigorous and systematic approach for monitoring and recording AE data in clinical settings that involve n-3 supplementation is required.
doi:10.1186/1471-2318-13-41
PMCID: PMC3664575  PMID: 23634646
Fish oil; Eicosapentaenoic acid; Docosahexaenoic acid; Adverse events; Older adults
23.  Structural features for α-galactomannan binding to galectin-1 
Glycobiology  2011;22(4):543-551.
Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-d-galacto-β(1 → 4)-d-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with 15N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-d-galactopyranosyl doublets surrounded by regions of about four or more “naked” mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [61-α(1 → 6)-d-galactosyl-β-d-mannotriaose and 63,64-di-α(1 → 6)-d-galactosyl-β-d-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.
doi:10.1093/glycob/cwr173
PMCID: PMC3287016  PMID: 22156919
galactose; glycan; lectin; NMR; protein
24.  High TCR Stimuli Prevent iTreg Differentiation in an NF-κB-Dependent Manner1 
The concentration of antigen or mitogenic stimuli is known to play an important role in controlling the differentiation of naïve CD4+ T cells into different effector phenotypes. In particular, whereas TCR engagement at low antigen doses in the presence of TGF-β and IL-2 can promote differentiation of Foxp3-expressing induced regulatory T cells (iTregs), high levels of antigen have been shown in vitro and in vivo to prevent Foxp3 upregulation. This tight control of iTreg differentiation dictated by antigen dose likely determines the quality and duration of an immune response. However, the molecular mechanism by which this high dose-inhibition of Foxp3 induction occurs is not well understood. In this study, we demonstrate that when cells are in the presence of CD28 costimulation, TCR-dependent NF-κB signaling is essential for Foxp3 inhibition at high doses of TCR engagement in mouse T cells. Prevention of Foxp3 induction depends on the production of NF-κB-dependent cytokines by the T cells themselves. Moreover, T cells that fail to upregulate Foxp3 under iTreg-differentiating conditions and high TCR stimulation acquire the capacity to make TNF and IFN-γ, as well as IL-17 and IL-9, especially if IFN-γ signaling is antagonized. Thus, NF-κB helps T cells control their differentiation fate in a cell-intrinsic manner and prevents peripheral iTreg development under conditions of high antigen load that may require more vigorous effector T cell responses.
doi:10.4049/jimmunol.1002361
PMCID: PMC3544303  PMID: 21411734
25.  Structural aspects of binding of α-linked digalactosides to human galectin-1 
Glycobiology  2011;21(12):1627-1641.
By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH–π stacking. These issues are resolved by a combination of 15N–1H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH–π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH–π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.
doi:10.1093/glycob/cwr083
PMCID: PMC3219418  PMID: 21712397
agglutinin; glycolipid; glycoprotein; lectin; sugar code

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