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1.  The dosage of Patz1 modulates reprogramming process 
Scientific Reports  2014;4:7519.
The acquisition of pluripotent cells can be achieved by combined overexpression of transcription factors Oct4, Klf4, Sox2 and c-Myc in somatic cells. This cellular reprogramming process overcomes various barriers to re-activate pluripotency genes and re-acquire the highly dynamic pluripotent chromatin status. Many genetic and epigenetic factors are essentially involved in the reprogramming process. We previously reported that Patz1 is required for maintenance of ES cell identity. Here we report that Patz1 plays an inhibitory role in OKSM-induced reprogramming process since more iPS colonies can be induced from Patz1+/− MEFs than wild type MEFs; while the addition of Patz1 significantly repressed reprogramming efficiency. Patz1+/− MEFs can surpass the senescence barrier of Ink4a/Arf locus, thus enhancing iPS colonies formation. Moreover, Patz1+/− MEFs displayed higher levels of acetylated histone H3, H3K4me2, H3K4me3, H3K36me3 and lower levels of histone H3K9me3 and HP1α, indicating that heterozygous knockout of Patz1 results in a globally open chromatin which is more accessible for transcriptional activation. However, Patz1−/− MEFs gave the lowest reprogramming efficiency which may result from cell senescence trigged by up-regulated Ink4a/Arf locus. Together, we have demonstrated that the dosage of Patz1 modulates reprogramming process via significantly influencing cell senescence, proliferation and chromatin structure.
doi:10.1038/srep07519
PMCID: PMC4268633  PMID: 25515777
2.  Observational study of upper gastrointestinal tract bleeding events in patients taking duloxetine and nonsteroidal anti-inflammatory drugs: a case-control analysis 
Purpose
To determine whether the concomitant use of duloxetine with prescription nonsteroidal anti-inflammatory drugs (NSAIDs) or aspirin was associated with an increased risk for upper gastrointestinal (UGI) bleeding compared with taking these analgesics alone.
Methods
Truven Health Analytics MarketScan Research Databases were examined for hospital admissions of adult patients indexed from January 1, 2007–December 31, 2011. Cases were patients with UGI hemorrhage or peptic ulcer disease. Controls were randomly selected from the remaining admissions to match 10:1 with cases based on age, sex, and admission date. Prescription medication exposure groups of interest were: 1) no exposure to duloxetine, NSAIDs or aspirin; 2) duloxetine only; 3) NSAIDs or aspirin only; 4) duloxetine plus NSAIDs or aspirin. Logistic regression and relative excess risk due to interaction was utilized to estimate any increased risk of UGI bleeding for patients prescribed these medications across these groups.
Results
There were 33,571 cases and 335,710 controls identified. Comparing exposure group 2 and group 4, the adjusted odds ratio was 1.03 (95% confidence interval [CI], 0.94, 1.12), and the adjusted relative excess risk due to interaction was 0.352 (95% CI: –0.18, 0.72) for risk of UGI bleeding, neither of which support an increased risk or an interaction between duloxetine and prescription NSAID or aspirin for these events.
Conclusion
There was no evidence of an increased risk for UGI bleeding when duloxetine was taken with prescription NSAIDs or aspirin. In addition, there was no evidence of an interaction between duloxetine and prescription NSAIDs or aspirin for an increased risk of these events.
doi:10.2147/DHPS.S66835
PMCID: PMC4222172  PMID: 25382984
duloxetine; upper gastrointestinal bleeding; NSAIDs; aspirin
3.  Epigenetic Landscapes Explain Partially Reprogrammed Cells and Identify Key Reprogramming Genes 
PLoS Computational Biology  2014;10(8):e1003734.
A common metaphor for describing development is a rugged “epigenetic landscape” where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from spin-glass physics. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells are a natural consequence of high-dimensional landscapes, and predicts that partially reprogrammed cells should be hybrids that co-express genes from multiple cell fates. We verify this prediction by reanalyzing existing datasets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity.
Author Summary
Traditionally, standard development has been viewed as a one-way process; an organism starts as a single cell (embryonic stem cell, ESC) that divides into a multitude of mature cell types (skin cells, heart, liver, etc). But, in 2006 Takahashi and Yamanaka revolutionized this view by stochastically converting skin cells into cell types resembling ESC (called induced pluripotent stem cells, iPSC). Following this groundbreaking experiment, other reprogramming protocols have been found, so now scientists can switch between a variety of cell types such as ESC, skin, liver, neurons, and heart. This has already revolutionized the understanding of biology and could change the future of medicine. A common metaphor for development is Waddington's landscape, in which an ESC is like a ball rolling down a hill which eventually ends in a valley (mature cell type). In this paper, we make this analogy more precise by developing a mathematical model of cellular development. Using data on real cell types, we can provide insight into existing reprogramming protocols and potentially predict new reprogramming protocols.
doi:10.1371/journal.pcbi.1003734
PMCID: PMC4133049  PMID: 25122086
4.  Solitary fibrous tumors of the pleura with Doege-Potter syndrome: a case report and three-decade review of the literature 
BMC Research Notes  2014;7(1):515.
Background
No case of solitary fibrous tumor of the pleura with Doege-Potter syndrome has been reported in China. This study was to report a rare repeatedly recurrent case of solitary fibrous tumor of the pleura with Doege-Potter syndrome diagnosed in China and a three-decade literature review of solitary fibrous tumor of the pleura with Doege-Potter syndrome worldwide.
Case presentation
A rare case of solitary fibrous tumor of the pleura with Doege-Potter syndrome was diagnosed in 2005 with follow-up to 2011. All medical records were collected and literature of solitary fibrous tumor of the pleura with Doege-Potter syndrome from 1979 to 2011 was obtained through Medline. This typical case, diagnosed and confirmed by histopathologic results, was a 72-year-old Chinese woman who had a complaint of night sweat for a month. A localized mass 12 cm × 11 cm × 8 cm in size was found associated with pleural effusion in her left low chest cavity, and blood tests showed severe hypoglycemia. Removal of the mass solved the hypoglycemia. The case was repeatedly recurrent in April, 2010 and March, 2011 and had no signs of recurrence up to the end of 2011 after surgery. A review of 45 cases of solitary fibrous tumor of the pleura with Doege-Potter syndrome compared and summarized clinical characteristics, treatments, and outcomes by benign and malignant tumor nature.
Conclusions
Incidence of solitary fibrous tumor of the pleura with Doege-Potter syndrome is similar between genders. There are no significant differences in clinical characteristics between benign and malignant cases. Surgery is the first effective treatment for solitary fibrous tumor of the pleura with Doege-Potter syndrome and the completeness of the initial resection is the key to preventing recurrence. Routine follow-up examinations are recommended for early detection of recurrence.
doi:10.1186/1756-0500-7-515
PMCID: PMC4267432  PMID: 25113505
Solitary fibrous tumors of pleura; Doege-Potter syndrome; Surgery; Case report review
5.  Next Generation Sequencing Analysis of Gene Regulation in the Rat Model of Retinopathy of Prematurity 
Purpose
To identify the genes, biochemical signaling pathways and biological themes involved in the pathogenesis of retinopathy of prematurity (ROP).
Methods
Next-generation sequencing (NGS) was performed on the RNA transcriptome of rats with the Penn et al. (1994) oxygen-induced retinopathy (OIR) model of ROP at the height of vascular abnormality, postnatal day (P) 19, and normalized to age-matched, room-air-reared littermate controls. Eight custom developed pathways with potential relevance to known ROP sequelae were evaluated for significant regulation in ROP: The three major Wnt signaling pathways, canonical, planar cell polarity (PCP), and Wnt/Ca2+, two signaling pathways mediated by the Rho GTPases RhoA and Cdc42, which are respectively thought to intersect with canonical and noncanonical Wnt signaling, nitric oxide signaling pathways mediated by two nitrox oxide synthase (NOS) enzymes, neuronal (nNOS) and endothelial (eNOS), and the retinoic acid (RA) signaling pathway. Regulation of other biological pathways and themes were detected by gene ontology using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the NIH's Database for Annotation, Visualization and Integrated Discovery (DAVID)'s GO terms databases.
Results
Canonical Wnt signaling was found to be regulated, but the non-canonical PCP and Wnt/Ca2+ pathways were not. Nitric oxide (NO) signaling, as measured by the activation of nNOS eNOS, was also regulated, as was RA signaling. Biological themes related to protein translation (ribosomes), neural signaling, inflammation and immunity, cell cycle and cell death, were (among others) highly regulated in ROP rats.
Conclusions
These several genes and pathways identified by NGS might provide novel targets for intervention in ROP.
doi:10.1007/s10633-013-9396-8
PMCID: PMC3775648  PMID: 23775346
6.  Gene Networks of Fully Connected Triads with Complete Auto-Activation Enable Multistability and Stepwise Stochastic Transitions 
PLoS ONE  2014;9(7):e102873.
Fully-connected triads (FCTs), such as the Oct4-Sox2-Nanog triad, have been implicated as recurring transcriptional motifs embedded within the regulatory networks that specify and maintain cellular states. To explore the possible connections between FCT topologies and cell fate determinations, we employed computational network screening to search all possible FCT topologies for multistability, a dynamic property that allows the rise of alternate regulatory states from the same transcriptional network. The search yielded a hierarchy of FCTs with various potentials for multistability, including several topologies capable of reaching eight distinct stable states. Our analyses suggested that complete auto-activation is an effective indicator for multistability, and, when gene expression noise was incorporated into the model, the networks were able to transit multiple states spontaneously. Different levels of stochasticity were found to either induce or disrupt random state transitioning with some transitions requiring layovers at one or more intermediate states. Using this framework we simulated a simplified model of induced pluripotency by including constitutive overexpression terms. The corresponding FCT showed random state transitioning from a terminal state to the pluripotent state, with the temporal distribution of this transition matching published experimental data. This work establishes a potential theoretical framework for understanding cell fate determinations by connecting conserved regulatory modules with network dynamics. Our results could also be employed experimentally, using established developmental transcription factors as seeds, to locate cell lineage specification networks by using auto-activation as a cipher.
doi:10.1371/journal.pone.0102873
PMCID: PMC4109943  PMID: 25057990
7.  Comparative Mitogenomics of Plant Bugs (Hemiptera: Miridae): Identifying the AGG Codon Reassignments between Serine and Lysine 
PLoS ONE  2014;9(7):e101375.
Insect mitochondrial genomes are very important to understand the molecular evolution as well as for phylogenetic and phylogeographic studies of the insects. The Miridae are the largest family of Heteroptera encompassing more than 11,000 described species and of great economic importance. For better understanding the diversity and the evolution of plant bugs, we sequence five new mitochondrial genomes and present the first comparative analysis of nine mitochondrial genomes of mirids available to date. Our result showed that gene content, gene arrangement, base composition and sequences of mitochondrial transcription termination factor were conserved in plant bugs. Intra-genus species shared more conserved genomic characteristics, such as nucleotide and amino acid composition of protein-coding genes, secondary structure and anticodon mutations of tRNAs, and non-coding sequences. Control region possessed several distinct characteristics, including: variable size, abundant tandem repetitions, and intra-genus conservation; and was useful in evolutionary and population genetic studies. The AGG codon reassignments were investigated between serine and lysine in the genera Adelphocoris and other cimicomorphans. Our analysis revealed correlated evolution between reassignments of the AGG codon and specific point mutations at the antidocons of tRNALys and tRNASer(AGN). Phylogenetic analysis indicated that mitochondrial genome sequences were useful in resolving family level relationship of Cimicomorpha. Comparative evolutionary analysis of plant bug mitochondrial genomes allowed the identification of previously neglected coding genes or non-coding regions as potential molecular markers. The finding of the AGG codon reassignments between serine and lysine indicated the parallel evolution of the genetic code in Hemiptera mitochondrial genomes.
doi:10.1371/journal.pone.0101375
PMCID: PMC4079613  PMID: 24988409
8.  Identification of Selective Small Molecule Inhibitors of the Nucleotide-Binding Oligomerization Domain 1 (NOD1) Signaling Pathway 
PLoS ONE  2014;9(5):e96737.
NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.
doi:10.1371/journal.pone.0096737
PMCID: PMC4013053  PMID: 24806487
9.  Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors 
Cell stem cell  2013;13(4):10.1016/j.stem.2013.09.002.
Summary
Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells.
doi:10.1016/j.stem.2013.09.002
PMCID: PMC3888026  PMID: 24094326
10.  Combined administration of testosterone plus an ornithine decarboxylase inhibitor as a selective prostate-sparing anabolic therapy 
Aging Cell  2013;13(2):303-310.
Because of its anabolic effects on muscle, testosterone is being explored as a function-promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterone’s adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterone’s promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth-promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone-responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α-difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterone’s effects on prostate, but did not affect testosterone’s anabolic effects on muscle. Co-administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate-sparing anabolic therapy.
doi:10.1111/acel.12174
PMCID: PMC4331775  PMID: 24305501
aging; anti-aging; sarcopenia; sex hormones; skeletal muscle; steroids
11.  Structure-guided optimization of small molecule c-Abl activators 
c-Abl kinase is maintained in its normal inactive state in the cell through an assembled, compact conformation. We describe two chemical series that bind to the myristoyl site of the c-Abl kinase domain and stimulate c-Abl activation. We hypothesize that these molecules activate c-Abl either by blocking the C-terminal helix from adopting a bent conformation that is critical for the formation of the autoinhibited conformation or by simply providing no stabilizing interactions to the bent conformation of this helix. Structure-based molecular modeling guided the optimization of binding and activation of c-Abl of these two chemical series and led to the discovery of c-Abl activators with nanomolar potency. The small molecule c-Abl activators reported herein could be used as molecular tools to investigate the biological functions of c-Abl and therapeutic implications of its activation.
Electronic supplementary material
The online version of this article (doi:10.1007/s10822-014-9731-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s10822-014-9731-5
PMCID: PMC3949015  PMID: 24573412
c-Abl; Myristoyl; Kinase activation; Kinase activators; Multi-fragment search; Docking
12.  Zfp322a Regulates Mouse ES Cell Pluripotency and Enhances Reprogramming Efficiency 
PLoS Genetics  2014;10(2):e1004038.
Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotency maintenance of mouse ES cells. Here, we report that Zfp322a is required for mES cell identity since depletion of Zfp322a directs mES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Pou5f1 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of mES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated mES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits mES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka's factors (OSKM). It can be even used in combination with Yamanaka's factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of mouse ES cells.
Author Summary
Embryonic stem (ES) cells are featured by their ability to self-renew and by their potential to differentiate into many different cell types. Recent studies have revealed that the unique properties of mouse ES cells are governed by a specific transcription regulatory network, including master regulators Oct4/Sox2/Nanog and other pluripotency factors. The importance of these factors was highlighted by the subsequent finding that combination of several transcription factors can reprogram differentiated fibroblasts back to pluripotent stem cells. Here, we report that Zfp322a is a novel factor which is required for mES cell identity. We revealed that Zfp322a can regulate the key pluripotency genes Pou5f1 and Nanog and functions as a repressor of MAPK/ERK pathway in mES cells, therefore preventing mES cell differentiation. Furthermore, we discovered that Zfp332a can promote the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Our results reveal that Zfp322a is a novel essential transcription factor which not only regulates ES cell pluripotency but also enhances iPSC formation.
doi:10.1371/journal.pgen.1004038
PMCID: PMC3923668  PMID: 24550733
13.  Central role for RNase YbeY in Hfq-dependent and Hfq-independent small-RNA regulation in bacteria 
BMC Genomics  2014;15:121.
Background
Conceptual parallels exist between bacterial and eukaryotic small-RNA (sRNA) pathways, yet relatively little is known about which protein may recognize and recruit bacterial sRNAs to interact with targets. In eukaryotes, Argonaute (AGO) proteins discharge such functions. The highly conserved bacterial YbeY RNase has structural similarities to the MID domain of AGOs. A limited study had indicated that in Sinorhizobium meliloti the YbeY ortholog regulates the accumulation of sRNAs as well as the target mRNAs, raising the possibility that YbeY may play a previously unrecognized role in bacterial sRNA regulation.
Results
We have applied a multipronged approach of loss-of-function studies, genome-wide mRNA and sRNA expression profiling, pathway analysis, target prediction, literature mining and network analysis to unravel YbeY-dependent molecular responses of E. coli exposed to hydroxyurea (HU). Loss of ybeY function, which results in a marked resistance to HU, had global affects on sRNA-mediated gene expression. Of 54 detectable E. coli sRNAs in our microarray analysis, 30 sRNAs showed a differential expression upon HU stress, of which 28 sRNAs displayed a YbeY-dependent change in expression. These included 12 Hfq-dependent and 16 Hfq-independent sRNAs. We successfully identified at least 57 experimentally inferred sRNA-mRNA relationships. Further applying a ‘context likelihood of relatedness’ algorithm, we reverse engineered the YbeY-dependent Hfq-dependent sRNA-mRNA network as well as YbeY-dependent Hfq-independent sRNA-mRNA network.
Conclusion
YbeY extensively modulates Hfq-dependent and independent sRNA-mRNA interactions. YbeY-dependent sRNAs have central roles in modulating cellular response to HU stress.
doi:10.1186/1471-2164-15-121
PMCID: PMC3933206  PMID: 24511998
YbeY; Small-RNA; Hfq; Hydroxyurea; Stress adaptation; E. coli
14.  First Record of the Leafhopper Genus Varicopsella Hamilton, 1980 (Hemiptera: Cicadellidae: Macropsinae) in China, with Descriptions of a New Subgenus and New Species, a Checklist, and a Key to Species 
A new monobasic leafhopper subgenus, Varicopsella (Multispinulosa) Li, Dai, and Li, subgen. nov., of the subfamily Macropsinae (Hemiptera: Auchenorrhyncha: Membracoidea: Cicadellidae) is proposed to accommodate Varicopsella (Multispinulosa) hamiltoni Li, Dai, and Li, sp. nov. from Guangxi province of China. The new subgenus and new species are described and illustrated. They can be distinguished mainly by characteristics of the fore wings with two anteapical cells; weak dorsoventrally flattened body; aedeagal shaft with paired apical processes on ventral margin; and the shape of the dorsal connective. An updated checklist and an illustrated key for identification of the species of Varicopsella along with geographical distributions of the species are given.
doi:10.1673/031.014.125
PMCID: PMC4222318  PMID: 25368069
Auchenorrhyncha; morphology; distribution
15.  Description of two new species of the leafhopper subgenus Pediopsoides (Pediopsoides) (Hemiptera, Cicadellidae, Macropsinae) from Guangxi Province, Southern China 
ZooKeys  2013;15-23.
Two new species of the Macropsinae leafhopper subgenus Pediopsoides (Pediopsoides) Matsumura, 1912, Pediopsoides (Pediopsoides) damingshanensis Li, Dai & Li, sp. n. and Pediopsoides (Pediopsoides) tishetshkini Li, Dai & Li sp. n., are described and illustrated from Guangxi Province of southern China. A key to males is provided to distinguish the species of the subgenus along with a map showing the distribution of the new species.
doi:10.3897/zookeys.321.5454
PMCID: PMC3744143  PMID: 23950687
Auchenorrhyncha; taxonomy; morphology; description
16.  Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway 
PLoS ONE  2013;8(8):e69619.
NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.
doi:10.1371/journal.pone.0069619
PMCID: PMC3731320  PMID: 23936340
17.  THE ANTI-AGING PROTEIN KLOTHO ENHANCES OLIGODENDROCYTE MATURATION AND MYELINATION OF THE CENTRAL NERVOUS SYSTEM 
We have previously shown that myelin abnormalities and loss characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. While the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. In the present study, we found significant effects of Klotho on oligodendrocyte functions including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein Western analysis indicated Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPCs maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knockout mice and their control littermates revealed that knockout mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knockout mice. Strikingly, at the ultrastructural level, Klotho knockout mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. In order to decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Taken together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes.
doi:10.1523/JNEUROSCI.2080-12.2013
PMCID: PMC3711388  PMID: 23365232
18.  The complete mitochondrial genomes of three parasitic nematodes of birds: a unique gene order and insights into nematode phylogeny 
BMC Genomics  2013;14:414.
Background
Analyses of mitochondrial (mt) genome sequences in recent years challenge the current working hypothesis of Nematoda phylogeny proposed from morphology, ecology and nuclear small subunit rRNA gene sequences, and raise the need to sequence additional mt genomes for a broad range of nematode lineages.
Results
We sequenced the complete mt genomes of three Ascaridia species (family Ascaridiidae) that infest chickens, pigeons and parrots, respectively. These three Ascaridia species have an identical arrangement of mt genes to each other but differ substantially from other nematodes. Phylogenetic analyses of the mt genome sequences of the Ascaridia species, together with 62 other nematode species, support the monophylies of seven high-level taxa of the phylum Nematoda: 1) the subclass Dorylaimia; 2) the orders Rhabditida, Trichinellida and Mermithida; 3) the suborder Rhabditina; and 4) the infraorders Spiruromorpha and Oxyuridomorpha. Analyses of mt genome sequences, however, reject the monophylies of the suborders Spirurina and Tylenchina, and the infraorders Rhabditomorpha, Panagrolaimomorpha and Tylenchomorpha. Monophyly of the infraorder Ascaridomorpha varies depending on the methods of phylogenetic analysis. The Ascaridomorpha was more closely related to the infraorders Rhabditomorpha and Diplogasteromorpha (suborder Rhabditina) than they were to the other two infraorders of the Spirurina: Oxyuridorpha and Spiruromorpha. The closer relationship among Ascaridomorpha, Rhabditomorpha and Diplogasteromorpha was also supported by a shared common pattern of mitochondrial gene arrangement.
Conclusions
Analyses of mitochondrial genome sequences and gene arrangement has provided novel insights into the phylogenetic relationships among several major lineages of nematodes. Many lineages of nematodes, however, are underrepresented or not represented in these analyses. Expanding taxon sampling is necessary for future phylogenetic studies of nematodes with mt genome sequences.
doi:10.1186/1471-2164-14-414
PMCID: PMC3693896  PMID: 23800363
Mitochondrial genome; Ascaridia; Nematode; Gene arrangement; Phylogeny
19.  Ancestral Gene Organization in the Mitochondrial Genome of Thyridosmylus langii (McLachlan, 1870) (Neuroptera: Osmylidae) and Implications for Lacewing Evolution 
PLoS ONE  2013;8(5):e62943.
The first complete mitochondrial genome of the lacewing family Osmylidae (Thyridosmylus langii (McLachlan, 1870)) (Neuroptera) was sequenced in this study. The genome is a circular molecule of 16,221 bp containing the typical 37 genes but is arranged in the same order as that of the putative ancestor of hexapod and lacks translocation of trnC as shared by all previously sequenced neuropteran mtDNAs. This reveals that trnC translocation does not represent an organizational synapomorphy in the mitochondrion for the entire Neuroptera clade. Comparative analysis of neuropteran tRNA genes reveals a relatively slow and conserved evolution of the mitochondrion throughout the order. Secondary structure models of the ribosomal RNA genes of T. langii largely agree with those proposed for other insect orders. Nevertheless, domain I of T. langii rrnL is consisted of nine helices rather than eight helices which is typical for neuropteran rrnL. Protein-coding genes have typical mitochondrial start codons, with the exception of COI, which uses the TCG start codon also found in Ithonidae and Chrysopidae. Like other neuropteran insects, the control region is the most AT-rich region and comparatively simple, with little evidence of conserved blocks or long tandem repeats. Considering the issues of base-compositional and branch length heterogeneity, we used a range of phylogenetic approaches to recover neuropteridan relationships and explored the effect of method choice on recovery of monophyly of Neuropterida: ((Neuroptera + Megaloptera) + Raphidioptera). The monophyly of Neuroptera and the more basal position of Osmylidae were also recovered by different datasets and phylogenetic methods.
doi:10.1371/journal.pone.0062943
PMCID: PMC3662673  PMID: 23717397
20.  Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA 
Cell stem cell  2010;7(5):618-630.
SUMMARY
Clinical application of induced pluripotent stem (iPS) cells is limited by the low efficiency of iPS derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-integrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling and regenerative medicine.
doi:10.1016/j.stem.2010.08.012
PMCID: PMC3656821  PMID: 20888316
21.  Genomic Approaches to Deconstruct Pluripotency 
Embryonic stem cells (ESCs) first derived from the inner cell mass of blastocyst-stage embryos have the unique capacity of indefinite self-renewal and potential to differentiate into all somatic cell types. Similar developmental potency can be achieved by reprogramming differentiated somatic cells into induced pluripotent stem cells (iPSCs). Both types of pluripotent stem cells provide great potential for fundamental studies of tissue differentiation, and hold promise for disease modeling, drug development, and regenerative medicine. Although much has been learned about the molecular mechanisms that underlie pluripotency in such cells, our understanding remains incomplete. A comprehensive understanding of ESCs and iPSCs requires the deconstruction of complex transcription regulatory networks, epigenetic mechanisms, and biochemical interactions critical for the maintenance of self-renewal and pluripotency. In this review, we will discuss recent advances gleaned from application of global “omics” techniques to dissect the molecular mechanisms that define the pluripotent state.
doi:10.1146/annurev-genom-082410-101506
PMCID: PMC3652340  PMID: 21801025
22.  The transcriptional landscape of hematopoietic stem cell ontogeny 
Cell stem cell  2012;11(5):701-714.
Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSC purified from >2500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSC, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource (http://hsc.hms.harvard.edu) will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.
doi:10.1016/j.stem.2012.07.018
PMCID: PMC3545475  PMID: 23122293
23.  Mitochondrial Genomes of Two Barklice, Psococerastis albimaculata and Longivalvus hyalospilus (Psocoptera: Psocomorpha): Contrasting Rates in Mitochondrial Gene Rearrangement between Major Lineages of Psocodea 
PLoS ONE  2013;8(4):e61685.
The superorder Psocodea has ∼10,000 described species in two orders: Psocoptera (barklice and booklice) and Phthiraptera (parasitic lice). One booklouse, Liposcelis bostrychophila and six species of parasitic lice have been sequenced for complete mitochondrial (mt) genomes; these seven species have the most rearranged mt genomes seen in insects. The mt genome of a barklouse, lepidopsocid sp., has also been sequenced and is much less rearranged than those of the booklouse and the parasitic lice. To further understand mt gene rearrangements in the Psocodea, we sequenced the mt genomes of two barklice, Psococerastis albimaculata and Longivalvus hyalospilus, the first representatives from the suborder Psocomorpha, which is the most species-rich suborder of the Psocodea. We found that these two barklice have the least rearranged mt genomes seen in the Psocodea to date: a protein-coding gene (nad3) and five tRNAs (trnN, trnS1, trnE, trnM and trnC) have translocated. Rearrangements of mt genes in these two barklice can be accounted for by two events of tandem duplication followed by random deletions. Phylogenetic analyses of the mt genome sequences support the view that Psocoptera is paraphyletic whereas Phthiraptera is monophyletic. The booklouse, L. bostrychophila (suborder Troctomorpha) is most closely related to the parasitic lice. The barklice (suborders Trogiomorpha and Psocomorpha) are closely related and form a monophyletic group. We conclude that mt gene rearrangement has been substantially faster in the lineage leading to the booklice and the parasitic lice than in the lineage leading to the barklice. Lifestyle change appears to be associated with the contrasting rates in mt gene rearrangements between the two lineages of the Psocodea.
doi:10.1371/journal.pone.0061685
PMCID: PMC3632521  PMID: 23630609
24.  Regulation of Physiologic Actions of LRRK2: Focus on Autophagy 
Neuro-Degenerative Diseases  2011;10(1-4):238-241.
Background
Mutations in LRRK2 are associated with familial and sporadic Parkinson's disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown. Objectives: To investigate mechanisms by which LRRK2 might cause PD.
Methods
We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing.
Results
Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes.
Conclusion
These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.
doi:10.1159/000332599
PMCID: PMC3363354  PMID: 22204929
LRRK2 mutations; Autophagy; Familial and sporadic Parkinson's disease
25.  Inverted Expression Profiles of Sex-Biased Genes in Response to Toxicant Perturbations and Diseases 
PLoS ONE  2013;8(2):e56668.
The influence of sex factor is widely recognized in various diseases, but its molecular basis, particularly how sex-biased genes, those with sexually dimorphic expression, behave in response to toxico-pathological changes is poorly understood. In this study, zebrafish toxicogenomic data and transcriptomic data from human pathological studies were analysed for the responses of male- and female-biased genes. Our analyses revealed obvious inverted expression profiles of sex-biased genes, where affected males tended to up-regulate genes of female-biased expression and down-regulate genes of male-biased expression, and vice versa in affected females, in a broad range of toxico-pathological conditions. Intriguingly, the extent of these inverted profiles correlated well to the susceptibility or severity of a given toxico-pathological state, suggesting that inverted expression profiles of sex-biased genes observed in this study can be used as important indicators to assess biological disorders.
doi:10.1371/journal.pone.0056668
PMCID: PMC3573008  PMID: 23457601

Results 1-25 (50)