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1.  Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition 
Cell  2013;155(5):1034-1048.
LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their lifecycles, whereas hosts have developed mechanisms to combat retrotransposition’s mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the co-purified proteome, identifying 37 high-confidence candidate interactors. These datasets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the novel findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest this occurs during or immediately after target-primed reverse transcription.
PMCID: PMC3904357  PMID: 24267889
2.  Mechanisms of U87 Astrocytoma Cell Uptake and Trafficking of Monomeric versus Protofibril Alzheimer’s Disease Amyloid-β Proteins 
PLoS ONE  2014;9(6):e99939.
A significant hallmark of Alzheimer’s disease is the formation of senile plaques in the brain due to the unbalanced levels of amyloid-beta (Aβ). However, although how Aβ is produced from amyloid precursor proteins is well understood, little is known regarding the clearance and metabolism of various Aβ aggregates from the brain. Similarly, little is known regarding how astrocytes internalize and degrade Aβ, although astrocytes are known to play an important role in plaque maintenance and Aβ clearance. The objective of this study is to investigate the cellular mechanisms that mediate the internalization of soluble monomeric versus oligomeric Aβ by astrocytes. We used a combination of laser confocal microscopy and genetic and pharmacological experiments to dissect the internalization of sAβ42 and oAβ42 and their postendocytic transport by U87 human brain astrocytoma cell line. Both Aβ42 species were internalized by U87 cells through fluid phase macropinocytosis, which required dynamin 2. Depleting LDL receptor-related protein 1 (LRP1) decreased sAβ42 uptake more significantly than that of oAβ42. We finally show that both Aβ42 species were rapidly transported to lysosomes through an endolytic pathway and subjected to proteolysis after internalization, which had no significant toxic effects to the U87 cells under relatively low concentrations. We propose that macropinocytic sAβ42 and oAβ42 uptake and their subsequent proteolytic degradation in astroglial cells is a significant mechanism underlying Aβ clearance from the extracellular milieu. Understanding the molecular events involved in astrocytic Aβ internalization may identify potential therapeutic targets for Alzheimer’s disease.
PMCID: PMC4062444  PMID: 24941200
3.  Active Transposition in Genomes 
Annual review of genetics  2012;46:651-675.
Transposons are DNA sequences capable of moving in genomes. Early evidence showed their accumulation in many species and suggested their continued activity in at least isolated organisms. In the past decade, with the development of various genomic technologies, it has become abundantly clear that ongoing activity is the rule rather than the exception. Active transposons of various classes are observed throughout plants and animals, including humans. They continue to create new insertions, have an enormous variety of structural and functional impact on genes and genomes, and play important roles in genome evolution. Transposon activities have been identified and measured by employing various strategies. Here, we summarize evidence of current transposon activity in various plant and animal genomes.
PMCID: PMC3612533  PMID: 23145912
somatic mutation; transposon; retrotransposon; polymorphism; genome dynamics; structural variation
4.  Chromatin “pre-pattern” and epigenetic modulation in the cell fate choice of liver over pancreas in the endoderm 
Nucleus  2012;3(2):150-154.
Understanding the basis for multipotency, whereby stem cells and other progenitors can differentiate into certain tissues and not others, provides insights into the mechanism of cell programming in development, homeostasis, and disease. We recently reported a screen of diverse chromatin marks to obtain clues about chromatin states in the multipotent embryonic endoderm. Genetic and pharmacologic tests of certain marks’ function demonstrated that the relevant chromatin modifying factors modulate the fate choice for liver or pancreas induction in the endoderm. The information about chromatin states from embryonic studies can be used to predict lineage-specific developmental potential and chromatin modifiers to enhance particular cell fate transitions from stem cells.
PMCID: PMC3383570  PMID: 22555599
Chromatin; competence; endoderm; histone marks; liver; pancreas
5.  The Presence of Open Dentinal Tubules Affects the Biological Properties of Dental Pulp Cells Ex Vivo 
Molecules and Cells  2011;31(1):65-71.
To investigate the effects of open dentinal tubules on the morphological and functional characteristics of dental pulp cells. Morphological changes in human dental pulp cells that were seeded onto dentin discs with open dentinal tubules were investigated on days 1, 2, 4, and 10 of culture using scanning electron microscopy and fluorescence microscopy. Samples collected on days 1, 3, 6, 8, and 10 of culture were evaluated for cell proliferation rate and alkaline phosphatase activity. Cultured human dental pulp cells developed a columnar or polygonal morphology and monopolar cytoplasmic processes that extended into the dentinal tubules. The cells formed a multilayer and secreted an extracellular matrix onto the cell surface. Scanning electron microscopy and fluorescence microscopy revealed polarized organization of odontoblasts. Cells seeded onto dentin discs proliferated minimally but showed high levels of ALP activity. Dental pulp cells seeded onto treated dentin discs develop an odontoblastlike phenotype, which may be a potential alternative for use in experimental research on dentinogenesis.
PMCID: PMC3906866  PMID: 21120627
dental pulp cells; dentin disc; dentinal tubules; differentiation; odontoblasts
6.  Noninvasive diffuse optical monitoring of head and neck tumor blood flow and oxygenation during radiation delivery 
Biomedical Optics Express  2012;3(2):259-272.
This study explored using a novel diffuse correlation spectroscopy (DCS) flow-oximeter to noninvasively monitor blood flow and oxygenation changes in head and neck tumors during radiation delivery. A fiber-optic probe connected to the DCS flow-oximeter was placed on the surface of the radiologically/clinically involved cervical lymph node. The DCS flow-oximeter in the treatment room was remotely operated by a computer in the control room. From the early measurements, abnormal signals were observed when the optical device was placed in close proximity to the radiation beams. Through phantom tests, the artifacts were shown to be caused by scattered x rays and consequentially avoided by moving the optical device away from the x-ray beams. Eleven patients with head and neck tumors were continually measured once a week over a treatment period of seven weeks, although there were some missing data due to the patient related events. Large inter-patient variations in tumor hemodynamic responses were observed during radiation delivery. A significant increase in tumor blood flow was observed at the first week of treatment, which may be a physiologic response to hypoxia created by radiation oxygen consumption. Only small and insignificant changes were found in tumor blood oxygenation, suggesting that oxygen utilizations in tumors during the short period of fractional radiation deliveries were either minimal or balanced by other effects such as blood flow regulation. Further investigations in a large patient population are needed to correlate the individual hemodynamic responses with the clinical outcomes for determining the prognostic value of optical measurements.
PMCID: PMC3269843  PMID: 22312579
(170.0170) Medical optics and biotechnology; (170.3660) Light propagation in tissues; (170.3880) Medical and biological imaging; (170.6480) Spectroscopy, speckle
7.  Chromatin “Pre-Pattern” and Histone Modifiers in a Fate Choice for Liver and Pancreas 
Science (New York, N.Y.)  2011;332(6032):963-966.
Transcriptionally silent genes can be marked by histone modifications and regulatory proteins that indicate the genes’ potential to be activated. Such marks have been identified in pluripotent cells, but it is unknown how such marks occur in descendant, multipotent embryonic cells that have restricted cell fate choices. We isolated mouse embryonic endoderm cells and assessed histone modifications at regulatory elements of silent genes that are activated upon liver or pancreas fate choices. We found that the liver and pancreas elements have distinct chromatin patterns. Furthermore, the histone acetyltransferase P300, recruited via BMP signaling, and the histone methyltransferase Ezh2 have modulatory roles in the fate choice. These studies reveal a functional “pre-pattern” of chromatin states within multipotent progenitors and potential targets to modulate cell fate induction.
PMCID: PMC3128430  PMID: 21596989
8.  Mobile Interspersed Repeats Are Major Structural Variants in the Human Genome 
Cell  2010;141(7):1171-1182.
Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chip's usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.
PMCID: PMC2943426  PMID: 20602999
9.  Influences of tissue absorption and scattering on diffuse correlation spectroscopy blood flow measurements 
Biomedical Optics Express  2011;2(7):1969-1985.
In this study we evaluate the influences of optical property assumptions on near-infrared diffuse correlation spectroscopy (DCS) flow index measurements. The optical properties, absorption coefficient (µa) and reduced scattering coefficient (µs′), are independently varied using liquid phantoms and measured concurrently with the flow index using a hybrid optical system combining a dual-wavelength DCS flow device with a commercial frequency-domain tissue-oximeter. DCS flow indices are calculated at two wavelengths (785 and 830 nm) using measured µa and µs′ or assumed constant µa and µs′. Inaccurate µs′ assumptions resulted in much greater flow index errors than inaccurate µa. Underestimated/overestimated µs′ from −35%/+175% lead to flow index errors of +110%/−80%, whereas underestimated/overestimated µa from −40%/+150% lead to −20%/+40%, regardless of the wavelengths used. Examination of a clinical study involving human head and neck tumors indicates up to +280% flow index errors resulted from inter-patient optical property variations. These findings suggest that studies involving significant µa and µs′ changes should concurrently measure flow index and optical properties for accurate extraction of blood flow information.
PMCID: PMC3130582  PMID: 21750773
(170.0170) Medical optics and biotechnology; (170.3660) Light propagation in tissues; (170.3880) Medical and biological imaging; (170.6480) Spectroscopy, speckle
10.  The epigenetic profile of immunoglobulin genes is dynamically regulated during B cell differentiation and is modulated by pre-BCR signaling* 
Antigen receptor loci poised for V(D)J rearrangement undergo germline transcription (GT) of unrearranged genes, and the accessible gene segments are associated with post-translational modifications (PTMs) on histones. Here, we performed a comprehensive analysis of the dynamic changes of 4 PTMs throughout B and T cell differentiation in freshly isolated ex vivo cells. Methylation of lysines 4 and 79 of histone H3, and acetylation of H3, demonstrated stage- and lineage-specificity, and were most pronounced at the J segments of loci poised for, or undergoing, rearrangement, except for dimethylation of H3K4, which was more equally distributed on V, D and J genes. Focusing on the IgL loci, we demonstrated there are no active PTMs in the absence of pre-BCR signaling. Kappa locus GT and PTMs on Jκ genes are rapidly induced following pre-BCR signaling in large pre-B cells. In contrast, the λ locus shows greatly delayed onset of GT and PTMs, which do not reach high levels until the immature B cell compartment, the stage at which receptor editing is initiated. Analysis of MiEκ−/− mice shows that this enhancer plays a key role in inducing not only GT but PTMs. Using an inducible pre-B cell line, we demonstrate that active PTMs on Jκ genes occur after GT is initiated, indicating that histone PTMs do not make the Jκ region accessible, but conversely, GT may play a role in adding PTMs. Our data indicate that the epigenetic profile of IgL genes is dramatically modulated by pre-BCR signaling and B cell differentiation status.
PMCID: PMC2804480  PMID: 19155482
Gene rearrangement; gene regulation; B cells
11.  Interleukin-6 polymorphisms modify the risk of periodontitis: a systematic review and meta-analysis 
Objective: To clarify the association of IL-6 polymorphisms and periodontitis, a meta-analysis of case-control studies and a systemic review were conducted. Material and methods: We performed a literature search using PubMed and Medline database to May 2009, with no restrictions. We also reviewed references from all retrieved articles. Six case-control studies involving 1 093 periodontitis cases and 574 controls were selected for meta-analysis to assess the purported associations between IL-6 polymorphisms and the risk of periodontitis. IL-6 −174 G/C and −572 C/G polymorphisms were included in the present meta-analysis, and the association between IL-6 −6331 T/C polymorphism and the risk of periodontitis was adequately reviewed as well. Results and conclusion: The present meta-analysis indicates that the IL-6 −174 G allele could not modify the risk of chronic periodontitis, but increased the risk of aggressive periodontitis. And −572 C/G polymorphism is associated with the pathogenesis of periodontitis, including chronic periodontitis or aggressive periodontitis.
PMCID: PMC2789527  PMID: 19946956
Interleukin-6; Polymorphism; Periodontitis; Risk; Meta-analysis
12.  Dental erosion and severe tooth decay related to soft drinks: a case report and literature review*  
Soft drinks have many potential health problems. The inherent acids and sugars have both acidogenic and cariogenic potential, resulting in dental caries and potential enamel erosion. In this report we present a 25-year-old man complaining with the severe worn-out of the front teeth during the past 3 years. He had a history of drinking cola for more than 7 years and had a poor oral hygiene. Severe decays were present in the incisors and the canines, while less severe lesions were noted on the premolars and the molars. The review is to show the relationship between dental erosion and caries and soft drinks. Some efforts have been taken to reduce the harmful effect of soft drinks.
PMCID: PMC2676420  PMID: 19434767
Dental erosion; Caries; Soft drinks; Toothbrushing
13.  Global Regulation of Nucleotide Biosynthetic Genes by c-Myc 
PLoS ONE  2008;3(7):e2722.
The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP) coupled with pair-end ditag sequencing analysis (ChIP-PET) revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated.
Methodology/Principal Findings
Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA) and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis.
Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.
PMCID: PMC2444028  PMID: 18628958
14.  Production of the Neurotoxin BMAA by a Marine Cyanobacterium 
Marine Drugs  2007;5(4):180-196.
Diverse species of cyanobacteria have recently been discovered to produce the neurotoxic non-protein amino acid β-methylamino-L-alanine (BMAA). In Guam, BMAA has been studied as a possible environmental toxin in the diets of indigenous Chamorro people known to have high levels of Amyotrophic Lateral Sclerosis/ Parkinsonism Dementia Complex (ALS/PDC). BMAA has been found to accumulate in brain tissues of patients with progressive neurodegenerative illness in North America. In Guam, BMAA was found to be produced by endosymbiotic cyanobacteria of the genus Nostoc which live in specialized cycad roots. We here report detection of BMAA in laboratory cultures of a free-living marine species of Nostoc. We successfully detected BMAA in this marine species of Nostoc with five different methods: HPLC-FD, UPLC-UV, Amino Acid Analyzer, LC/MS, and Triple Quadrupole LC/MS/MS. This consensus of five different analytical methods unequivocally demonstrates the presence of BMAA in this marine cyanobacterium. Since protein-associated BMAA can accumulate in increasing levels within food chains, it is possible that biomagnification of BMAA could occur in marine ecosystems similar to the biomagnification of BMAA in terrestrial ecosystems. Production of BMAA by marine cyanobacteria may represent another route of human exposure to BMAA. Since BMAA at low concentrations causes the death of motor neurons, low levels of BMAA exposure may trigger motor neuron disease in genetically vulnerable individuals.
PMCID: PMC2365698  PMID: 18463731
Nostoc; motor neuron disease; ALS/PDC; LC/MS/MS; biomagnification

Results 1-14 (14)