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1.  Plasma Biomarker of Dietary Phytosterol Intake 
PLoS ONE  2015;10(2):e0116912.
Dietary phytosterols, plant sterols structurally similar to cholesterol, reduce intestinal cholesterol absorption and have many other potentially beneficial biological effects in humans. Due to limited information on phytosterol levels in foods, however, it is difficult to quantify habitual dietary phytosterol intake (DPI). Therefore, we sought to identify a plasma biomarker of DPI.
Methods and Findings
Data were analyzed from two feeding studies with a total of 38 subjects during 94 dietary periods. DPI was carefully controlled at low, intermediate, and high levels. Plasma levels of phytosterols and cholesterol metabolites were assessed at the end of each diet period. Based on simple ordinary least squares regression analysis, the best biomarker for DPI was the ratio of plasma campesterol to the endogenous cholesterol metabolite 5-α-cholestanol (R2 = 0.785, P < 0.0001). Plasma campesterol and 5-α-cholestanol levels varied greatly among subjects at the same DPI level, but were positively correlated at each DPI level in both studies (r > 0.600; P < 0.01).
The ratio of plasma campesterol to the coordinately regulated endogenous cholesterol metabolite 5-α-cholestanol is a biomarker of dietary phytosterol intake. Conversely, plasma phytosterol levels alone are not ideal biomarkers of DPI because they are confounded by large inter-individual variation in absorption and turnover of non-cholesterol sterols. Further work is needed to assess the relation between non-cholesterol sterol metabolism and associated cholesterol transport in the genesis of coronary heart disease.
PMCID: PMC4323197  PMID: 25668184
2.  Insulin Resistance Is an Important Risk Factor for Cognitive Impairment in Elderly Patients with Primary Hypertension 
Yonsei Medical Journal  2014;56(1):89-94.
Insulin resistance plays a role in the development of dementia and hypertension. We investigated a possible relationship between cognitive impairment and insulin resistance in elderly Chinese patients with primary hypertension.
Materials and Methods
One hundred and thirty-two hypertensive elderly patients (>60 years) were enrolled in this study, and assigned into either the cognitive impairment group (n=61) or the normal cognitive group (n=71). Gender, age, education, body mass index (BMI), waist hip ratio (WHR), total cholesterol (TC), triglyceride (TG), C-reactive protein (CRP), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), creatinine (Cr), fasting plasma glucose (FPG), fasting insulin (FINS), homeostasis model of assessment for insulin resistance index (HOMA-IR), systolic blood pressure, diastolic blood pressure, smoking history, atherosclerosis and the proportion of uncontrolled hypertension were compared between the two groups. Multi-factorial logistic regression analysis was performed.
No significant differences were found in gender, age, TC, CRP, HDL-C, LDL-C, Cr, BP, smoking history, atherosclerosis and the proportion of uncontrolled hypertension between the two groups. The cognitive impairment group had lower education levels, and higher BMI, WHR, TG, FPG, FINS, and HOMA-IR levels than the control group. Logistic regression analysis revealed the levels of education, BMI, WHR, and HOMA-IR as independent factors that predict cognitive impairment in patients.
Our study demonstrates that poor education and increased BMI, WHR, and HOMA-IR are independent risk factors for cognitive impairment in elderly patients with hypertension. Insulin resistance plays an important role in the development of cognitive impairment in primary elderly hypertensive patients.
PMCID: PMC4276782  PMID: 25510751
Hypertension; insulin resistance; cognitive impairment; elderly; risk factor
3.  Decreased Cognitive Function in Extended Family Members from the Single Late-Onset-Alzheimer’s-Disease Pedigree 
A family history of dementia is associated with an increased risk of developing Alzheimer’s disease (AD) late in life (LOAD). This study marked the first attempt to assess the familial contribution to differences in cognitive performance in a large family-based group in the Chinese community. We enrolled 168 participants without dementia from a single pedigree with 9 probable AD patients diagnosed after age 65. These participants were evaluated with a comprehensive neuropsychological battery, the Chinese version of the Mini Mental State Examination, and the Alzheimer Disease Assessment Scale–Cognitive Subscale. Analyses found that extended family members of the LOAD pedigree showed similar performance on measures of global cognitive function and semantic memory compared to controls, but lower scores on episodic memory, attention, and executive function measures. These results indicate that the genetic influences on certain sub-cognitive domains are more detectable despite normal global cognitive function, and that family members with the LOAD pedigree are at risk for developing LOAD by virtue of their family history with an additive risk due to increased age. The findings in this study support the importance of documenting if there is a positive family history of AD in clinical evaluations.
PMCID: PMC3734807  PMID: 23742872
Alzheimer’s disease; Dementia; Genetics; Memory; Geriatric; Pedigree
4.  Translational selection in human: more pronounced in housekeeping genes 
Biology Direct  2014;9:17.
Translational selection is a ubiquitous and significant mechanism to regulate protein expression in prokaryotes and unicellular eukaryotes. Recent evidence has shown that translational selection is weakly operative in highly expressed genes in human and other vertebrates. However, it remains unclear whether translational selection acts differentially on human genes depending on their expression patterns.
Here we report that human housekeeping (HK) genes that are strictly defined as genes that are expressed ubiquitously and consistently in most or all tissues, are under stronger translational selection.
These observations clearly show that translational selection is also closely associated with expression pattern. Our results suggest that human HK genes are more efficiently and/or accurately translated into proteins, which will inevitably open up a new understanding of HK genes and the regulation of gene expression.
This article was reviewed by Yuan Yuan, Baylor College of Medicine; Han Liang, University of Texas MD Anderson Cancer Center (nominated by Dr Laura Landweber) Eugene Koonin, NCBI, NLM, NIH, United States of America Sandor Pongor, International Centre for Genetic Engineering and biotechnology (ICGEB), Italy.
PMCID: PMC4100034  PMID: 25011537
Translational selection; Codon usage bias; Expression regulation; Housekeeping gene; Tissue-specific gene
5.  A Simple Spatial Working Memory and Attention Test on Paired Symbols Shows Developmental Deficits in Schizophrenia Patients 
Neural Plasticity  2013;2013:130642.
People with neuropsychiatric disorders such as schizophrenia often display deficits in spatial working memory and attention. Evaluating working memory and attention in schizophrenia patients is usually based on traditional tasks and the interviewer's judgment. We developed a simple Spatial Working Memory and Attention Test on Paired Symbols (SWAPS). It takes only several minutes to complete, comprising 101 trials for each subject. In this study, we tested 72 schizophrenia patients and 188 healthy volunteers in China. In a healthy control group with ages ranging from 12 to 60, the efficiency score (accuracy divided by reaction time) reached a peak in the 20–27 age range and then declined with increasing age. Importantly, schizophrenia patients failed to display this developmental trend in the same age range and adults had significant deficits compared to the control group. Our data suggests that this simple Spatial Working Memory and Attention Test on Paired Symbols can be a useful tool for studies of spatial working memory and attention in neuropsychiatric disorders.
PMCID: PMC3856212  PMID: 24349797
6.  RiceWiki: a wiki-based database for community curation of rice genes 
Nucleic Acids Research  2013;42(Database issue):D1222-D1228.
Rice is the most important staple food for a large part of the world’s human population and also a key model organism for biological studies of crops as well as other related plants. Here we present RiceWiki (, a wiki-based, publicly editable and open-content platform for community curation of rice genes. Most existing related biological databases are based on expert curation; with the exponentially exploding volume of rice knowledge and other relevant data, however, expert curation becomes increasingly laborious and time-consuming to keep knowledge up-to-date, accurate and comprehensive, struggling with the flood of data and requiring a large number of people getting involved in rice knowledge curation. Unlike extant relevant databases, RiceWiki features harnessing collective intelligence in community curation of rice genes, quantifying users' contributions in each curated gene and providing explicit authorship for each contributor in any given gene, with the aim to exploit the full potential of the scientific community for rice knowledge curation. Based on community curation, RiceWiki bears the potential to make it possible to build a rice encyclopedia by and for the scientific community that harnesses community intelligence for collaborative knowledge curation, covers all aspects of biological knowledge and keeps evolving with novel knowledge.
PMCID: PMC3964990  PMID: 24136999
7.  Myb promotes centriole amplification and later steps of the multiciliogenesis program 
Development (Cambridge, England)  2013;140(20):4277-4286.
The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.
PMCID: PMC3787764  PMID: 24048590
Myb; Multiciliogenesis; Centriole amplification
8.  Ginkgolide B Reduces LOX-1 Expression by Inhibiting Akt Phosphorylation and Increasing Sirt1 Expression in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells 
PLoS ONE  2013;8(9):e74769.
Oxidized low-density lipoprotein (ox-LDL) is an important risk factor in the development of atherosclerosis. LOX-1, a lectin-like receptor for ox-LDL, is present primarily on endothelial cells and upregulated by ox-LDL, tumor necrosis factor a, shear stress, and cytokines in atherosclerosis. Recent studies demonstrated that ginkgolide B, a platelet-activating factor receptor antagonist, has antiinflammatory and antioxidant effects on endothelial and nerve cells. The present study investigated the effects of ginkgolide B on LOX-1 expression and the possible mechanism of action. Our results showed that ginkgolide B inhibited LOX-1 and intercellular cell adhesion molecule-1 (ICAM-1) expression in ox-LDL-stimulated endothelial cells through a mechanism associated with the attenuation of Akt activation. Similar data were obtained by silencing Akt and LY294002. We also evaluated Sirt1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. These molecules play a protective role in endothelial cell injury. The results showed that ginkgolide B increased Sirt1 expression in ox-LDL-treated cells. The inhibitory effects of ginkgolide B on LOX-1 and ICAM-1 expression were reduced in Sirt1 siRNA-transfected cells. Nrf2 expression was increased in ox-LDL-treated cells, and ginkgolide B downregulated Nrf2 expression. These results suggest that ginkgolide B reduces Nrf2 expression by inhibiting LOX-1 expression, consequently reducing oxidative stress injury in ox-LDL-stimulated cells. Altogether, these results indicate that the protective effect of ginkgolide B on endothelial cells may be attributable to a decrease in LOX-1 expression and an increase in Sirt1 expression in ox-LDL-stimulated endothelial cells, the mechanism of which is linked to the inhibition of Akt activation. Ginkgolide B may be a multiple-target drug that exerts protective effects in ox-LDL-treated human umbilical vein endothelial cells.
PMCID: PMC3775799  PMID: 24069345
9.  d-chiro-Inositol is absorbed but not synthesized in rodents 
The British journal of nutrition  2009;102(10):1426-1434.
d-chiro-Inositol (DCI) and pinitol (1D-3-O-methyl-chiro-inositol) are distinctive inositols reported to possess insulin-mimetic properties. DCI-containing compounds were abundant in common laboratory animal feed. By GC-MS of 6 M-HCl hydrolysates, Purina Laboratory Rodent Diet 5001 (diet 5001) contained 0.23% total DCI by weight with most found in the Lucerne and soy meal components. In contrast, only traces of l-chiro-inositol were observed. The DCI moiety was present in a water-soluble non-ionic form of which most was shown to be pinitol. To measure the absorption of dietary inositols, rats were fed diet 5001 in a balance study or given purified pinitol or [2H6]DCI. More than 98% of the total DCI fed to rats as diet 5001, purified pinitol or [2H6]DCI was absorbed from the gastrointestinal tract. Rats chronically on diet 5001 consumed 921 μmol total DCI/kg body weight pet d but excreted less than 5.3% in the stool and urine, suggesting that the bulk was metabolized. The levels of pinitol or DCI in plasma, stool, or urine remained relatively stable in mice fed Purina PicoLab® Rodent Diet 20 5053 over a 5-week period, whereas these values declined to very low levels in mice fed a pinitol/DCI-deficient chemically-defined diet. To test whether DCI was synthesized or converted from myo-inositol, mice were treated with heavy water or [2H6]myo-inositol. DCI was neither synthesized endogenously from 2H-labelled water nor converted from [2H6]myo-inositol. DCI and pinitol in rodents appear to be derived solely from the diet.
PMCID: PMC3760478  PMID: 19586572
Pinitol; Insulin; Mass spectrometry; Defined diet
10.  Human Sodium/Inositol Cotransporter 2 (SMIT2) Transports Inositols But Not Glucose in L6 Cells 
To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [3H]D-chiro-inositol (DCI) by 159-fold. [3H]myo-Inositol uptake increased by 37-fold. In contrast, [14C]D-glucose, [14C]2-deoxy-D-glucose, or [14C]3-O-methyl-D-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The Km of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a Ki of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 hours) increased [3H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.
PMCID: PMC3732106  PMID: 19032932
SMIT2; D-chiro-inositol; myo-inositol; insulin resistance; diabetes; PCOS; DCI; glucose; pinitol
11.  The Effects of Phytosterols Present in Natural Food Matrices on Cholesterol Metabolism and LDL-Cholesterol: A Controlled Feeding Trial 
European journal of clinical nutrition  2010;64(12):1481-1487.
Extrinsic phytosterols supplemented to the diet reduce intestinal cholesterol absorption and plasma LDL-cholesterol. However, little is known about their effects on cholesterol metabolism when given in native, unpurified form and in amounts achievable in the diet. The objective of this investigation was to test the hypothesis that intrinsic phytosterols present in unmodified foods alter whole-body cholesterol metabolism.
Twenty out of 24 subjects completed a randomized, crossover feeding trial where all meals were provided by a metabolic kitchen. Each subject consumed two diets for 4 weeks each. The diets differed in phytosterol content (phytosterol-poor diet, 126 mg phytosterols/2000 kcal; phytosterol-abundant diet, 449 mg/2000 kcal) but were otherwise matched for nutrient content. Cholesterol absorption and excretion were determined by gas chromatograph/mass spectrometry after oral administration of stable isotopic tracers.
The phytosterol-abundant diet resulted in lower cholesterol absorption [54.2 ± 2.2 % (95% confidence interval, 50.5%, 57.9%) vs. 73.2 ± 1.3% (69.5%, 76.9%), P<0.0001] and 79% higher fecal cholesterol excretion [1322 ± 112 (1083.2, 1483.3) vs. 739 ± 97 mg/day (530.1, 930.2), P<0.0001] relative to the phytosterol-poor diet. Plasma lathosterol/cholesterol ratio rose 82% [from 0.71 ± 0.11 (0.41, 0.96) to 1.29 ± 0.14 μg/mg (0.98, 1.53), (P<0.0001)]. LDL-cholesterol was similar between diets.
Intrinsic phytosterols at levels present in a healthy diet are biologically active and have large effects on whole body cholesterol metabolism not reflected in circulating LDL. More work is needed to assess the effects of phytosterol-mediated fecal cholesterol excretion on coronary heart disease risk in humans.
PMCID: PMC3715129  PMID: 20808333
Diets; Absorption; Mass Spectrometry; Deuterium
12.  Nanoparticle-Based Systems for T1-Weighted Magnetic Resonance Imaging Contrast Agents 
Because magnetic resonance imaging (MRI) contrast agents play a vital role in diagnosing diseases, demand for new MRI contrast agents, with an enhanced sensitivity and advanced functionalities, is very high. During the past decade, various inorganic nanoparticles have been used as MRI contrast agents due to their unique properties, such as large surface area, easy surface functionalization, excellent contrasting effect, and other size-dependent properties. This review provides an overview of recent progress in the development of nanoparticle-based T1-weighted MRI contrast agents. The chemical synthesis of the nanoparticle-based contrast agents and their potential applications were discussed and summarized. In addition, the recent development in nanoparticle-based multimodal contrast agents including T1-weighted MRI/computed X-ray tomography (CT) and T1-weighted MRI/optical were also described, since nanoparticles may curtail the shortcomings of single mode contrast agents in diagnostic and clinical settings by synergistically incorporating functionality.
PMCID: PMC3676856  PMID: 23698781
T1-weighted magnetic resonance; molecular imaging; nanoparticles; contrast agents
13.  On the classification of long non-coding RNAs 
RNA Biology  2013;10(6):924-933.
Long non-coding RNAs (lncRNAs) have been found to perform various functions in a wide variety of important biological processes. To make easier interpretation of lncRNA functionality and conduct deep mining on these transcribed sequences, it is convenient to classify lncRNAs into different groups. Here, we summarize classification methods of lncRNAs according to their four major features, namely, genomic location and context, effect exerted on DNA sequences, mechanism of functioning and their targeting mechanism. In combination with the presently available function annotations, we explore potential relationships between different classification categories, and generalize and compare biological features of different lncRNAs within each category. Finally, we present our view on potential further studies. We believe that the classifications of lncRNAs as indicated above are of fundamental importance for lncRNA studies, helpful for further investigation of specific lncRNAs, for formulation of new hypothesis based on different features of lncRNA and for exploration of the underlying lncRNA functional mechanisms.
PMCID: PMC4111732  PMID: 23696037
long non-coding RNA; lncRNA; lncRNA classification; RNA transcripts
14.  Embelin-induced MCF-7 breast cancer cell apoptosis and blockade of MCF-7 cells in the G2/M phase via the mitochondrial pathway 
Oncology Letters  2012;5(3):1005-1009.
Embelin is a small molecular inhibitor extracted from Myrsinaceae plants that specifically inhibits XIAP, affecting the proliferation and apoptosis of various types of tumor cells. In our previous studies, we have demonstrated that embelin is able to induce the apoptosis of MCF-7 breast cancer cells in a dose-dependent manner. However, its mechanism of action is not yet clear. The purpose of this study was to investigate the involvement of the mitochondrial pathway in embelin-induced apoptosis and the effect of embelin on the cell cycle. Different doses of embelin were added to MCF-7 breast cancer cells and it was found that embelin was able to induce apoptosis of MCF-7 breast cancer cells in a dose- and time-dependent manner. Flow cytometry analysis revealed that embelin caused changes in the MCF-7 cell mitochondrial membrane potential and blocked the cell cycle of MCF-7 cells in the G2/M phase. Moreover, embelin was demonstrated to promote mitochondrial release of cytochrome C via regulation of Bax and Bcl-2, resulting in the activation of caspase-3 and -9, while no significant changes in the level of caspase-8 were observed. The results have demonstrated that embelin-induced apoptosis of MCF-7 breast cancer cells involves the mitochondrial pathway.
PMCID: PMC3576316  PMID: 23425971
embelin; XIAP; breast cancer MCF-7 cell; apoptosis; mitochondria
15.  Combined Effects of Ezetimibe and Phytosterols on Cholesterol Metabolism: A Randomized, Controlled Feeding Study in Humans 
Circulation  2011;124(5):596-601.
Both ezetimibe and phytosterols inhibit cholesterol absorption. We tested the hypothesis that ezetimibe combined with phytosterols is more effective than ezetimibe alone in altering cholesterol metabolism.
Methods and Results
Twenty-one mildly hypercholesterolemic subjects completed a randomized, double-blind, placebo-controlled, triple crossover study. Each subject received a phytosterol-controlled diet plus (1) ezetimibe placebo + phytosterol placebo, (2) 10 mg ezetimibe/day + phytosterol placebo, and (3) 10 mg ezetimibe/day + 2.5 g phytosterols/day, for 3 weeks each. All meals were prepared in a metabolic kitchen. Primary outcomes were intestinal cholesterol absorption, fecal cholesterol excretion, and LDL cholesterol levels. The combined treatment resulted in significantly lower intestinal cholesterol absorption (598 mg/day, 95% CI 368 to 828) relative to control (2161 mg/day, 1112 to 3209) and ezetimibe alone (1054 mg/day, 546 to 1561, both P < 0.0001). Fecal cholesterol excretion was significantly greater (P < 0.0001) with combined treatment (962 mg/day, 757 to 1168) relative to control (505 mg/day, 386 to 625) and ezetimibe alone (794 mg/day, 615 to 973). Plasma LDL cholesterol values during control, ezetimibe alone, and ezetimibe + phytosterols averaged 129 (95% CI: 116 to 142), 108 (97 to 119), and 101 (90 to 112) mg/dL (P < 0.0001 relative to control).
The addition of phytosterols to ezetimibe significantly enhanced the effects of ezetimibe on whole-body cholesterol metabolism and plasma LDL cholesterol. The large cumulative action of combined dietary and pharmacologic treatment on cholesterol metabolism emphasizes the potential importance of dietary phytosterols as adjunctive therapy for the treatment of hypercholesterolemia.
PMCID: PMC3304455  PMID: 21768544
Cholesterol synthesis/absorption/cardiovascular diseases; Randomized controlled clinical trial; Diet; Stable isotopes; Mass spectrometry
16.  Glycosidic Bond Cleavage is Not Required for Phytosteryl Glycoside-Induced Reduction of Cholesterol Absorption in Mice 
Lipids  2011;46(8):701-708.
Phytosteryl glycosides occur in natural foods but little is known about their metabolism and bioactivity. Purified acylated steryl glycosides (ASG) were compared with phytosteryl esters (PSE) in mice. Animals on a phytosterol-free diet received ASG or PSE by gavage in purified soybean oil along with tracers cholesterol-d7 and sitostanol-d4. In a three-day fecal recovery study, ASG reduced cholesterol absorption efficiency by 45 ± 6% compared with 40 ± 6% observed with PSE. Four hours after gavage, plasma and liver cholesterol-d7 levels were reduced 86% or more when ASG was present. Liver total phytosterols were unchanged after ASG administration but were significantly increased after PSE. After ASG treatment both ASG and deacylated steryl glycosides (SG) were found in the gut mucosa and lumen. ASG was quantitatively recovered from stool samples as SG. These results demonstrate that ASG reduces cholesterol absorption in mice as efficiently as PSE while having little systemic absorption itself. Cleavage of the glycosidic linkage is not required for biological activity of ASG. Phytosteryl glycosides should be included in measurements of bioactive phytosterols.
PMCID: PMC3314266  PMID: 21538209
Stable isotopes; Plant sterols; Mass spectrometry; Lipid absorption
17.  Interactions Between the Kinetochore Complex and the Protein Kinase A Pathway in Saccharomyces cerevisiae 
G3: Genes|Genomes|Genetics  2012;2(7):831-841.
The kinetochore is a large structure composed of multiple protein subcomplexes that connect chromosomes to spindle microtubules to enable accurate chromosome segregation. Significant advances have been made in the identification of kinetochore proteins and elucidation of kinetochore structure; however, comparatively little is known about how cellular signals integrate with kinetochore function. In the budding yeast Saccharomyces cerevisiae, the cyclic AMP protein kinase A signaling pathway promotes cellular growth in response to glucose. In this study, we find that decreasing protein kinase A activity, either by overexpressing negative regulators of the pathway or deleting the upstream effector Ras2, improves the viability of ipl1 and spc24 kinetochore mutants. Ipl1/Aurora B is a highly conserved kinase that corrects attachment of sister kinetochores that have attached to the same spindle pole, whereas Spc24 is a component of the conserved Ndc80 kinetochore complex that attaches directly to microtubules. Unexpectedly, we find that kinetochore mutants have increased phosphorylation levels of protein kinase A substrates, suggesting that the cyclic AMP protein kinase A signaling pathway is stimulated. The increase in protein kinase A activity in kinetochore mutants is not induced by activation of the spindle checkpoint or a metaphase delay because protein kinase A activity remains constant during an unperturbed cell cycle. Finally, we show that lowering protein kinase A activity can rescue the chromosome loss defect of the inner kinetochore ndc10 mutant. Overall, our data suggest that the increased protein kinase A activity in kinetochore mutants is detrimental to cellular growth and chromosome transmission fidelity.
PMCID: PMC3385989  PMID: 22870406
budding yeast; protein kinase A; kinetochore; chromosome segregation; spindle checkpoint
18.  Plasticity of DNA methylation in mouse T cell activation and differentiation 
BMC Molecular Biology  2012;13:16.
Circulating CD4+ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4+ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.
Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.
We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.
PMCID: PMC3386888  PMID: 22642378
DNA demethylation; T cell activation; T cell differentiation; Il2, Csf2
19.  An Integrated Analysis of miRNA and mRNA Expressions in Non-Small Cell Lung Cancers 
PLoS ONE  2011;6(10):e26502.
Using DNA microarrays, we generated both mRNA and miRNA expression data from 6 non-small cell lung cancer (NSCLC) tissues and their matching normal control from adjacent tissues to identify potential miRNA markers for diagnostics. We demonstrated that hsa-miR-96 is significantly and consistently up-regulated in all 6 NSCLCs. We validated this result in an independent set of 35 paired tumors and their adjacent normal tissues, as well as their sera that are collected before surgical resection or chemotherapy, and the results suggested that hsa-miR-96 may play an important role in NSCLC development and has great potential to be used as a noninvasive marker for diagnosing NSCLC. We predicted potential miRNA target mRNAs based on different methods (TargetScan and miRanda). Further classification of miRNA regulated genes based on their relationship with miRNAs revealed that hsa-miR-96 and certain other miRNAs tend to down-regulate their target mRNAs in NSCLC development, which have expression levels permissive to direct interaction between miRNAs and their target mRNAs. In addition, we identified a significant correlation of miRNA regulation with genes coincide with high density of CpG islands, which suggests that miRNA may represent a primary regulatory mechanism governing basic cellular functions and cell differentiations, and such mechanism may be complementary to DNA methylation in repressing or activating gene expression.
PMCID: PMC3203153  PMID: 22046296
20.  Dilatory is a Drosophila protein related to AZI1 (CEP131) that is located at the ciliary base and required for cilium formation 
Journal of Cell Science  2011;124(15):2622-2630.
A significant number of ciliary disease genes have been found to encode proteins that localise to the basal body. By contrast, a large number of basal-body-associated proteins remain to be characterised. Here, we report the identification of a new basal body protein that is required for ciliogenesis in Drosophila. Dilatory (DILA) is a predicted coiled-coil protein homologous to vertebrate AZI1 (also known as CEP131). Mutations in dila specifically exhibit defects in ciliated cells (sensory neurons and sperm). Several features of the neuronal phenotype suggest a defect in intraflagellar transport. In sensory neuron cilia, DILA protein localises to the ciliary base, including the basal body and putative transition zone, and it interacts genetically with the ciliary coiled-coil protein, Uncoordinated. These data implicate DILA in regulating intraflagellar transport at the base of sensory cilia.
PMCID: PMC3138703  PMID: 21750193
Cilia; Drosophila; Basal body; Sensory neuron; Spermatozoa
21.  Analysis of synonymous codon usage in Hepatitis A virus 
Virology Journal  2011;8:174.
Hepatitis A virus is the causative agent of type A viral hepatitis, which causes occasional acute hepatitis. Nevertheless, little information about synonymous codon usage pattern of HAV genome in the process of its evolution is available. In this study, the key genetic determinants of codon usage in HAV were examined.
The overall extent of codon usage bias in HAV is high in Picornaviridae. And the patterns of synonymous codon usage are quite different in HAV genomes from different location. The base composition is closely correlated with codon usage bias. Furthermore, the most important determinant that results in such a high codon bias in HAV is mutation pressure rather than natural selection.
HAV presents a higher codon usage bias than other members of Picornaviridae. Compositional constraint is a significant element that influences the variation of synonymous codon usage in HAV genome. Besides, mutation pressure is supposed to be the major factor shaping the hyperendemic codon usage pattern of HAV.
PMCID: PMC3087699  PMID: 21496278
22.  The Gene Regulatory Cascade Linking Proneural Specification with Differentiation in Drosophila Sensory Neurons 
PLoS Biology  2011;9(1):e1000568.
Temporal expression profiling of sensory precursor cells reveals how the atonal proneural transcription factor regulates a specialized neuronal differentiation pathway.
In neurogenesis, neural cell fate specification is generally triggered by proneural transcription factors. Whilst the role of proneural factors in fate specification is well studied, the link between neural specification and the cellular pathways that ultimately must be activated to construct specialised neurons is usually obscure. High-resolution temporal profiling of gene expression reveals the events downstream of atonal proneural gene function during the development of Drosophila chordotonal (mechanosensory) neurons. Among other findings, this reveals the onset of expression of genes required for construction of the ciliary dendrite, a key specialisation of mechanosensory neurons. We determine that atonal activates this cellular differentiation pathway in several ways. Firstly, atonal directly regulates Rfx, a well-known highly conserved ciliogenesis transcriptional regulator. Unexpectedly, differences in Rfx regulation by proneural factors may underlie variations in ciliary dendrite specialisation in different sensory neuronal lineages. In contrast, fd3F encodes a novel forkhead family transcription factor that is exclusively expressed in differentiating chordotonal neurons. fd3F regulates genes required for specialized aspects of chordotonal dendrite physiology. In addition to these intermediate transcriptional regulators, we show that atonal directly regulates a novel gene, dilatory, that is directly associated with ciliogenesis during neuronal differentiation. Our analysis demonstrates how early cell fate specification factors can regulate structural and physiological differentiation of neuronal cell types. It also suggests a model for how subtype differentiation in different neuronal lineages may be regulated by different proneural factors. In addition, it provides a paradigm for how transcriptional regulation may modulate the ciliogenesis pathway to give rise to structurally and functionally specialised ciliary dendrites.
Author Summary
Early during development, cells differentiate and take on specialized forms and functions. This requires the activation of specific genes for different cellular pathways. Our study addresses how this activation is regulated in the developing Drosophila nervous system. In this model, it is well known that proneural transcription factors are involved in directing cells to differentiate into various types of neurons. However, the mechanism by which they choreograph the activation of genes for neuronal differentiation is not clear. In this study, we focused on events leading to differentiation of mechanosensory neurons, which have specialized dendritic processes that mediate sensory perception. In these developing neurons we profiled the time course of gene expression that is triggered by the proneural factor atonal. Our analysis revealed the activation of genes required for the formation of these specialized dendrites, called cilia. We then identified several ways in which atonal regulated these genes. First, it activates intermediate transcription factors that regulate different subsets of differentiation genes. Second, in at least one case, atonal activates a differentiation gene directly, one that is involved in the formation of cilia (ciliogenesis). These findings offer new insight into how proneural factors regulate specialized neuronal differentiation pathways.
PMCID: PMC3023811  PMID: 21283833
23.  Phytosterol-deficient and high-phytosterol diets developed for controlled feeding studies 
Phytosterols reduce cholesterol absorption and low-density lipoprotein (LDL) cholesterol concentrations, but the quantity and physiological significance of phytosterols in common diets are generally unknown because nutrient databases do not contain comprehensive phytosterol data. The primary aim of this study was to design prototype phytosterol-deficient and high-phytosterol diets for use in controlled feeding studies of the influence of phytosterols on health. A second aim was to quantify the phytosterol content of these prototype diets and three other diets consumed in the United States. This study was conducted from June, 2001 to September, 2008 and involved designing, preparing, and then analyzing five different diets: an experimental phytosterol-deficient ‘control’ diet, a relatively high-phytosterol diet based on the Dietary Approaches to Stop Hypertension (DASH) diet, American Heart Association (AHA) diet, Atkins® lifetime maintenance plan, and a vegan diet. A single day of meals for each diet was homogenized and the resulting composites were analyzed for free, esterified, and glycosylated phytosterols by gas chromatography. Independent samples t tests were used to compare the diets’ total phytosterol content. The total phytosterol content of the experimental phytosterol-deficient diet was 64 mg/2000 kcal, with progressively larger quantities in Atkins®, AHA, vegan, and the high-phytosterol DASH diet (163, 340, 445 and 500 mg/2000 kcal, respectively). Glycosylated phytosterols, which are often excluded from phytosterol analyses, comprised 15.9 ± 5.9% (mean±SD) of total phytosterols. In summary, phytosterol-deficient and high-phytosterol diets that conform to recommended macronutrient guidelines and are palatable can now be used in controlled feeding studies.
PMCID: PMC2833354  PMID: 19942022
cholesterol; glycosylated phytosterol; DASH diet
24.  Defining the chromatin signature of inducible genes in T cells 
Genome Biology  2009;10(10):R107.
Inducible genes in T cells show the chromatin characteristics of active genes, suggesting they are primed for transcription.
Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation.
To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation.
These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation.
PMCID: PMC2784322  PMID: 19807913
25.  A novel DNA sequence periodicity decodes nucleosome positioning 
Nucleic Acids Research  2008;36(19):6228-6236.
There have been two types of well-characterized DNA sequence periodicities; both are found to be associated with important molecular mechanisms. One is a 3-nt periodicity corresponding to codon triplets, the other is a 10.5-nt periodicity related to the structure of DNA helixes. In the process of analyzing the genome and transcriptome of Trichomonas vaginalis, we observed a 120.9-nt periodicity along DNA sequences. Different from the 3- and 10.5-nt periodicities, this novel periodicity originates near the 5′-end of transcripts, extends along the direction of transcription, and weakens gradually along transcripts. As a result, codon usage as well as amino acid composition is constrained by this periodicity. Similar periodicities were also identified in other organisms, but with variable length associated with the length of nucleosome units. We validated this association experimentally in T. vaginalis, and demonstrated that the periodicity manifests nucleotide variations between linker-DNA and wrapping-DNA along nucleosome array. We conclude that this novel DNA sequence periodicity is a signature of nucleosome organization suggesting that nucleosomes are well-positioned with regularity, especially near the 5′-end of transcripts.
PMCID: PMC2577358  PMID: 18829715

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